Advances in Parasitology Volume 44

Advances in PARASITOLOGY VOLUME 44 Editorial Board C. Bryant Division of Biochemistry and Molecular Biology, The Aus...

Author: John R. Baker | Ralph Muller | David Rollinson

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Advances in PARASITOLOGY

VOLUME 44

Editorial Board C. Bryant Division of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT 0200, Australia

M. Coluzzi Director, Istituto di Parassitologia, Universita Degli Studi di Roma ‘La Sapienza’, P. le A. Moro 5, 00185 Roma, Italy C. Combes Laboratoire de Biologie Animale, Universitt de Perpignan, Centre de Biologie et d’Ecologie Tropicale et MCditerranCenne, Avenue de Villeneuve, 66860 Perpignan Cedex, France

D.D. Despommier Division of Tropical Medicine and Environmental Sciences, Department of Microbiology, Columbia University, 630 West 168‘h Street, New York, NY 10032, USA W.H.R. Lumsden 16A Merchiston Crescent, Edinburgh, EHlO 5AX, UK J.J. Shaw Instituto de Ciincias Biomtdicas, Universidade de Siio Paulo, av. Prof. Lineu Prestes 1374,05508-900, Cidade Universitaria, Siio Paulo, SP, Brazil

Lord Soulsby of Swaflham Prior Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 OES, UK

K. Tanabe Laboratory of Biology, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-Ku, Osaka 535, Japan

P. Wenk Falkenweg 69, D-72076 Tubingen, Germany

Advances in PARASIT0LOGY Edited by

J.R. BAKER Royal Society of Tropical Medicine and Hygiene, London, England

R. MULLER International Institute of Parasitology, St Albans, England and

D. ROLLINSON The Natural History Museum, London, England VOLUME 44

ACADEMIC PRESS A Harcourt Science and Technology Company

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CONTRIBUTORS TO VOLUME 44 N . BOULTER,Department of Biology, PO Box 373, University of York, York YO1 5 Y W , U K L A . CHISHOLM, Department of Parasitology, The University of Queensland, Brisbane, Queensland 4072, Australia E. HANDMAN,The Walter and Eliza Hall Institute of Medical Research, Post Ofice Royal Melbourne Hospital, Victoria 3050, Australia R. HULL, Department of Biology, PO Box 373, University of York, York YO1 5 Y W , UK H . MONE, Laboratoire de Biologie Animale, U M R no. 5555 du CNRS, Centre de Biologie et d'Ecologie tropicale et mhditerrane'enne, Universith, Avenue de Villeneuve, 66860 Perpignan Cedex, France S. MORAND, Laboratoire de Biologie Animale, U M R no. 5555 du C N R S , Centre de Biologie et d'Ecologie tropicale et me'diterrane'enne, Universite', Avenue de Villeneuve, 66860 Perpignan Cedex, France G. MOUAHID,Laboratoire de Biologie Animale, U M R no. 5555 du C N R S , Centre de Biologie et d'Ecologie tropicale et me'diterranhenne, Universite', Avenue de Villeneuve, 66860 Perpignan Cedex, France A.W. PIKE, Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, and Marine Harvest McConnell, Lochailort, Inverness-shire PH38 4 L Z , U K K . ROHDE,School of Biological Sciences, Division of Zoology, University of New South Wales 2351, Australia S.L. WADSWORTH,Department of Zoology, University of Aberdeen. Tillydrone Avenue, Aberdeen AB24 2 T Z , and Marine Harvest McConnell, Lochailort, Inverness-shire PH38 4 L Z , UK I.D. WHITTINGTON, Department of Parasitology, The University of Queensland, Brisbane, Queensland 4072, Australia

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The volume opens with a review of the cell biology of the flagellate protozoan genus Leishmania by Emanuela Handman (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia). Infection with any of about ten species of this genus in humans causes one of three main clinical manifestations: cutaneous, mucocutaneous or visceral leishmaniasis. The World Health Organization estimates that there are currently from 3 to 5 million cases in the world and the prevalence is rising. Visceral leishmaniasis is usually fatal if not treated and is reckoned to have killed about 75 000 people in one year alone in recent outbreaks in India and Sudan. The author reviews recent progress made in understanding the cell biology of this fascinating parasite, which invades macrophages, the very cells which should protect against invading organisms, as it shuttles between the mammalian tissues and the gut of the sandfly intermediate host. The host and parasite molecules that facilitate the establishment of infection, parasite survival in the two hosts and its transmission from one to the other are given special emphasis. She forecasts that the complete genome will be sequenced in the coming decade, and the challenge will be to identify the function of the genes and then to understand the whole organism better. The volume continues with an account by Nicola Boulter and Roger Hall (University of York, UK) of the immune response of cattle to the apicomplexan protozoan parasites Theileria annulata, T. parva and T. sergenti. The global cost of these parasites to agriculture is estimated to be over one billion (lo9) US dollars annually. Immunity to T. annulata and T. parva is predominantly cell mediated (by cytostatic macrophages and cytotoxic lymphocytes, respectively), while that against T. sergenti, although less understood, appears to be mainly humoral. Control of these infections is currently directed against the parasites, by chemotherapy or vaccination, and against the vectors (ticks). Neither the use of acaricides nor chemotherapy is particularly effective, and both are expensive. Vaccination against T. annulata gives over 90% homologous protection and is often cross-protective against the other two species also. However, it involves the use of a live, attenuated vaccine with the attendant need for constant refrigeration and the risk of inadvertent transfer of other pathogens with the vaccine. Current research is directed towards the production of effective,

viii

PREFACE

stable and cheap subunit vaccines requiring only a single application, and the authors predict that success will eventually be achieved with ‘naked DNA’ vaccines containing cytokine genes as immunopotentiators. Ian Whittington and Leslie Chisholm (University of Queensland, Australia) and Klaus Rohde (University of New England, Australia) have contributed the first detailed review of the larvae (oncomiracidia) of the class Monogenea for over 30 years (Advances in Parasitology 1, 1963, and 6, 1968). Members of this group have direct life cycles and some are economically important parasites of fishes, particularly in aquaculture. The authors have examined in detail the general morphology and behaviour of many examples, and also the structure of the epidermis, ciliated cells, haptorial sclerites, glands, digestive system, protonephridia and sense organs in particular. In 1957 a new classification of the group based on larval rather than adult characteristics was proposed and it was realized that the phylogenetic relationship to the Digenea was more remote than previously thought. Recently, there has been controversy about whether the subclasses Monopisthocotylea and Polyopisthocotylea have a monophyletic origin or whether the many similar characteristics are due to convergence brought about by similar selection pressures. The authors have not been able to elucidate their phylogenetic relationships conclusively but indicate which larval characters, in addition to adult features and molecular data, must be combined to provide a comprehensive data set. They also point out many areas where there is still a lack of knowledge and this should act as a stimulus to further studies. The distribution of schistosomiasis reflects in part the distribution of potential intermediate host species. A knowledge of the intermediate snail hosts of schistosomes is essential for the recognition of transmission foci and allows an assessment of the risk of the disease spreading to new areas. In this review article Helene Mone, Gabriel Mouahid and Serge Morand (University of Perpignan, France) examine the distribution of Schistosoma bovis and consider the reported interactions occurring between parasites and snails. This species has a wide intermediate host spectrum and naturally infected molluscs belong to two genera, Planorbarius and Bulinus. The authors recognize three major groups of S . bovis populations (Iberian, Mediterranean and south Sahara) and propose a possible local adaptation to the parasite in the Iberian Peninsula. The article provides fresh insights into the biogeography of S. bovis and complements the recent review in Volume 41 by Jan De Bont and Jozef Vercruysse on schistosomiasis in cattle. The increasing demand for fresh salmon has given rise to a dramatic increase in high-density farming of fish in cages in Scotland, Norway and North America. Parasitologists are well aware that large groupings of hosts inevitably lead to disease problems and one of the most serious parasitic

PREFACE

ix

diseases in salmonid aquaculture is due to crustacean ectoparasites known as sealice. In 1998 the costs resulting from sealice damage in Scotland alone were estimated at &15-30 million. Not surprisingly there has been a considerable increase in research concerning sealice. In this detailed review on both biology and control, Alan Pike (University of Aberdeen, UK) and Simon Wadsworth (Marine Harvest McConnell, UK) pay particular attention to Lepeophtheirus salmonis and Caligus elongatus, the two common species of sealice. Much recent research has been directed towards finding new treatments and methods of control, and this review emphasizes the need to understand the basic biology of the parasite and identifies those research areas in need of further investigation. J.R. Baker R. Muller D. Rollinson


CONTRIBUTORS TO VOLUME 44 . . . . . . . . . . . . . . . . . PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

vii

Biology of Leishmania E. Handman Abstract . . . . . .

. . . . . . . . . . . . . .. . . . . . . . . . . . . . .. 2 . The Interaction of Leishmania with the Macrophage . . 3. The Interaction of Leishmania with the Sandfly . . . . . 4. Concluding Remarks . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . 1 . Introduction

. . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 5 21

26 21 21

Immunity and Vaccine Development in the Bovine Theilerioses N. Boulter and R . Hall

1. 2. 3. 4. 5. 6.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Theileria annulata . . . . . . . . . . . . . . . . . . . . . . . Theileriaparva . . . . . . . . . . . . . . . . . . . . . . . . Theileria sergenti . . . . . . . . . . . . . . . . . . . . . . . Comparative Aspects . . . . . . . . . . . . . . . . . . . . . TheFuture . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

42 43 51

61 11

80 82 83 83

xii

CONTENTS

The Distribution of Schistosoma bowis Sonsino. 1876 in Relation to Intermediate Host- Parasite Relationships H. Mone. G . Mouahid and S. Morand 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Collection of Data . . . . . . . . . . . . . . . . . . . . . . The Natural Mollusc Intermediate Host Spectrum . . . . . . Geographical Distributions of the Mollusc Intermediate Hosts Geographical Distribution of S . bovis . . . . . . . . . . . . The Experimental Mollusc Intermediate Host Spectrum . . . Compatibility in the Mollusc-S . bovis Association . . . . . . Three Main Populations of S . bovis . . . . . . . . . . . . . Paleobiogeographical Scenario of S . bovis . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

100 100 102 . 113 . 113 . 124 . 125 . 125 . 128 . 130 132 133 133

The Larvae of Monogenea (Platyhelminthes)

.

I.D. Whittington. L.A. Chisholm and K Rohde

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . General Morphology . . . . . . . . . . . . . . . . . . . . . Haptoral Sclerites. . . . . . . . . . . . . . . . . . . . . . . Ciliated Cells . . . . . . . . . . . . . . . . . . . . . . . . . Epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . Terminal Globule . . . . . . . . . . . . . . . . . . . . . . . Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protonephridia . . . . . . . . . . . . . . . . . . . . . . . . Sense Organs . . . . . . . . . . . . . . . . . . . . . . . . . Nervous System . . . . . . . . . . . . . . . . . . . . . . . Digestive Tract . . . . . . . . . . . . . . . . . . . . . . . . Parenchyma . . . . . . . . . . . . . . . . . . . . . . . . . Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

140 141 142 146 153 159 163 169 177 183 198 200 201 203 215 218 218

xiii

CONTENTS

Sealice on Salmonids: Their Biology and Control A.W. Pike and S.L. Wadsworth 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Species, Morphology, Host Range and Geographical Distribution The Reproductive System and Reproduction . . . . . . . . . Life Cycles of Sealice. . . . . . . . . . . . . . . . . . . . . Epidemiology of Sealice Infections . . . . . . . . . . . . . . Physiology of Sealice . . . . . . . . . . . . . . . . . . . . . Pathological Effects of Sealice on Salmonids . . . . . . . . . Treatment and Control of Infection . . . . . . . . . . . . . . Economics of Sealice Infection . . . . . . . . . . . . . . . . Priority Areas for Future Sealice Research . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF VOLUMES IN

THISSERIES . . . . . . . . . . . . .

234 234 238 245 261 268 279 286 292 311 317 318 318 339 349


Cell Biology of Leishmania Emanuela Handman

The Walter and Eliza Hall Institute of Medical Research. Post Ofice Royal Melbourne Hospital. Victoria 3050. Australia Abstract ....................................................................... 2 1. Introduction ................................................................. 2 2 . The Interaction of Leishmania with the Macrophage ........................... 5 2.1. Promastigote Invasion and Establishment of Infection ..................... 5 2.2. Promastigote to Amastigote Differentiation in the Macrophage ........... 15 2.3. Mechanisms of Parasite Persistence ..................................... 18 3. The Interaction of Leishmania with the Sandfly ............................... 21 3.1. Parasite Differentiation in the Blood Meal ................................ 21 3.2. Establishment of Infection in the Sandfly ................................ 22 3.3. Metacyclogenesis ...................................................... 23 3.4. Transmission to the Mammalian Host ................................... 24 4. Concluding Remarks........................................................ 26 4.1. Cell Biology in the Post-genome Era .................................... 26 Acknowledgements ........................................................... 27 References.................................................................... 27

ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

Copvrrghr 2000 Academic Press A / / righrs of rrprodurrron in ang fonn rr.wrvd

2

E. HANDMAN

ABSTRACT

Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly, where they grow as motile flagellated promastigotes in the gut, and the mammalian macrophage, where they survive and grow intracellularly as non-flagellated amastigotes in the phagolysosome. Leishmaniasis is the outcome of an evolutionary ‘arms race’ between the host’s immune system and the parasite’s evasion mechanisms, which ensure survival and transmission in the population. The diverse spectrum of patterns and severity of disease reflect the varying contributions of parasite virulence factors and host responses, some of which act in a host protective manner while others exacerbate disease. This chapter describes the interaction of the Leishmania with their hosts, with emphasis on the molecules and mechanisms evolved by the parasites to avoid, subvert or exploit the environments in the sandfly and the macrophage, and to move from one to the other.

1. INTRODUCTION

Leishmania parasites were first noticed by Cunningham in 1885, and described subsequently by Leishman in 1900 and Donovan in 1903 (quoted in Peters, 1988). Leishmaniasis is endemic in the tropical regions of Africa and the Americas, in the Indian subcontinent, and in the Mediterranean and South-west Asian regions. It is a group of diseases with a spectrum of clinical manifestations ranging from self-healing cutaneous ulcers to severe disease with massive tissue destruction and even death. Despite the varied clinical manifestations and the homing of the organisms to different organs, all leishmaniases are caused by infection with the protozoan parasite Leishmania. Moreover, all infections start with the introduction of the organisms into the skin by the bite of an infected sandfly. However, there is considerable diversity within the genus, with at least ten species of Leishmania that are pathogenic for humans. Different Leishmania species also display significant preference, if not absolute specificity, for particular sandfly species. Traditionally, leishmaniasis has been classified into three groups according to the clinical manifestations of disease. Cutaneous leishmaniasis is, by and large, a self-limiting, but chronic skin ulcer developing at the site of the sandfly bite, which may take months to heal. Mucocutaneous leishmaniasis initially causes similar skin ulcers that heal, but subsequently lesions reappear, primarily in the mucous tissue of the nose and mouth.

CELL BIOLOGY OF LEISHMANIA

3

These are often accompanied by secondary infections and massive tissue destruction. Visceral leishmaniasis is a very severe systemic disease, with the organisms homing to the liver, spleen and bone marrow. Visceral leishmaniasis is usually fatal if not treated. The global number of infected individuals cannot be determined with accuracy, but the World Health Organization (WHO) estimates that there are at least 3-5 million clinical cases among the 12 million infected individuals from a total population of about 350 million living in endemic areas (Modabber, 1993). Of the 1.5 million new cases each year, it is estimated that 500 000 are visceral leishmaniasis. The most recent epidemics of visceral leishmaniasis in India and the Sudan are estimated to have killed about 75 000 people in 1991 alone. Currently, a new wave of epidemic visceral leishmaniasis is sweeping through many parts of the world (McGregor, 1998). The general prevalence of leishmaniasis has increased significantly over the last decade or so, as a result of wars, environmental degradation and unplanned urbanization. Another significant development in leishmaniasis has been the reactivation of subclinical asymptomatic infection into full-blown disease in acquired immunodeficiency syndrome (AIDS) patients (Altges et al., 1991; WHO, 1995). All species of Leishmania are transmitted by sandfly vectors, either of the genus Phlebotomus (in the Old World) or Lutzomyia (in the New World), and it is generally accepted that all Leishmania are obligatory intracellular parasites in mammalian macrophages (Alexander and Russell, 1992) (Figure 1). The factors that determine the varied clinical manifestations and severity of leishmaniasis have been an area of intense study and much speculation. It is clear that the species of parasite initiating the infection is important, but equally important is the genetic susceptibility of both the insect vector and mammalian host (Convit et al., 1972; Wu and Tesh, 1990a; Liew and O'Donnell, 1993; Blackwell, 1996). Control of leishmaniasis has been hampered by the fact that the disease is primarily a zoonosis with large reservoirs of rodents, dogs or other animals. In addition, it is now apparent that asymptomatic infection is quite common and represents a potential reservoir in its own right (Aebischer, 1994). Organic pentavalent antimonials have formed the mainstay of treatment for the last half century (Pentostam, Wellcome or Glucantime, Rhone Poulenc). Other drugs such as amphotericin B, allopurinol and aminosidine (paromomycin) have not proved as useful as initially hoped (Olliaro and Bryceson, 1993). Control through vaccination, although the most costeffective form of disease eradication and a long-term quest of the WHOTDR Programme, has only been attempted for cutaneous leishmaniasis (Handman, 1997). Significant progress has been achieved in this area but

4

E. HANDMAN Intracellular amaatlaote " Transtormatlo

Mammalian host

Transformation

Proliferation in the midgut

Figure I A schematic representation of the Leishmania life cycle in the mammalian host and sandfly vector.

there is still a long way to go before production of a successful vaccine for mass administration in the field (WHO, 1998). Leishmaniasis, like many infectious diseases, is the aftermath of a protracted evolutionary 'arms race' between the host defence mechanisms and the parasite virulence factors. The host has evolved an innate rapid


5

deployment defence system, such as the complement system, which is not organism-specific and can be activated without delay. On the other hand, the parasite has developed strategies to overcome the innate immune system, and in so doing it can exploit the very system whose function it is to eliminate the parasites. In this chapter, a review of the progress made in understanding the cell biology of the Leishmania parasite as it shuttles between the sandfly gut and the mammalian tissues is presented. Special emphasis will be given to host and parasite molecules that facilitate the establishment of infection, the parasite survival in the two environments and its transmission from one host to the other.

2. THE INTERACTION OF LHSHMANlA WITH THE MACROPHAGE 2.1. Promastigote Invasion and Establishment of Infection

2.1.1. Promastigote Entry into the Mammalian Host As mentioned earlier, Leishmania are digenetic organisms shuttling between a flagellated promastigote, living in the midgut and foregut of the female sandfly, and an intracellular amastigote in the mammalian macrophage (Figure I). Sandflies generate a small pool of blood by the secretion of saliva into the wound from which they feed (Schlein, 1993). Leishmania promastigotes are deposited by the sandfly into this pool of blood. By analogy to the end-stage developmental forms of African trypanosomes, the developmental stage of the parasite introduced into the mammalian host has been named ‘metacyclic’ and the dividing, immature form present in the fly has been named ‘procyclic’ (Sacks, 1989). A detailed description of the process of metacyclogenesis and the structural changes involved are presented in Section 3.3. The first hurdle the promastigotes encounter in the new environment is the need to escape the lytic effects of serum complement. While the complement system has a central role in host defences against many microorganisms, pathogenic microbes have evolved mechanisms to evade it and, in some cases, such as in Leishmania, to exploit it (Figure 2). There are three mechanisms which activate the complement cascade: the classical pathway, which is primarily activated by immune complexes; the alternative pathway, which is activated by direct binding of the complement component C3 to the microbe surface; and the lectin pathway, which is initiated by binding of the mannose-binding protein to terminal mannose residues on microbial surfaces (Figure 2).

6

E. HANDMAN

Mannan-binding lectin pathway

Pathogen surfaces

binds mannose on pathogen surface

-

I

~3conveflase

I

~5 convertase

I

Membrane-attack complex

Figure 2 The main pathways and components of the complement activation system. (Adapted from Taylor, P. et al., 1998, Current Biology 8, R259-261.)

Early in vivo studies suggested that most promastigotes introduced into the host are killed rapidly and, until recently, killing was presumed to be via the activation of the alternative pathway of complement (Zuckerman, 1975; Alexander and Russell, 1992). Subsequent in vitro studies showed that promastigotes do indeed activate the alternative pathway (Mosser and Edelson, 1984). It was shown that the infectious stage of promastigotes, the metacyclic promastigotes, are much more resistant to lysis than the immature procyclic organisms despite the fact that both forms bind


7

significant amounts of C3b (Joiner, 1988; Puentes et al., 1988). Resistance to complement appears to be due to the inability of the membrane attack complex to penetrate the dense phosphoglycan coat on the parasite surface (Puentes et al., 1989, 1990). A major contributor to the resistance of metacyclic promastigotes to complement is the proteolytic activity of a membrane protease, the ‘leishmanolysin’ or gp63, which cleaves C3b to a form that cannot fix the membrane attack complex (Brittingham et al., 1995; Brittingham and Mosser, 1996). An added advantage to the parasite from the hydrolysis of C3b is the generation of the chemotactic peptides C3a and C5a, which attract monocytes to the area (Brittingham and Mosser, 1996). Newly arrived monocytes that are low in major histocompatibility complex (MHC) class I1 molecules cannot present antigen and are quiescent hosts for the parasites during the early phase of lesion formation (Murray, 1994). A totally new perspective on the mechanism of host cell invasion by promastigotes has been provided recently by Dominguez and Torano (1999), who showed that, in humans, the classical complement pathway plays a much more important role than the alternative pathway. In an in vitro system, promastigotes were shown: to bind natural IgM antibodies present in human blood; to attach to complement receptor CR1 on erythrocytes within seconds of contact with the blood; and to invade neutrophils, where they are destroyed, and also macrophages, where they survive (Dominguez and Torano, 1999). 2.1.2. Phagocytosis of Promastigotes by Macrophages Phagocytosis is an important effector mechanism for the eradication of micro-organisms, and is performed by ‘professional phagocytes’ such as polymorphonuclear cells and macrophages. Paradoxically, the macrophage is both the home of the parasite and also the means of its destruction. Phagocytosis transports the microbes into a cellular compartment where they can be killed and degraded. The macrophage also signals the presence of the intracellular microbe to cells of the adaptive immune system which can in turn activate the macrophage to destroy the parasites through mechanisms involving, in part, signalling via the receptor for y-interferon. Many microbes, including Mycobacterium tuberculosis and Leishmania, have developed mechanisms to subvert the macrophage microbicidal activity and have made it their preferred host cell. Macrophages therefore act both as host cells for the invading parasite and as antigen presenting cells to immune T cells, turning on the Th 1, macrophage-activating responses necessary for parasite destruction (Mauel, 1996). Macrophages are the final

8

E. HANDMAN

effector cells which kill the intracellular organisms once a protective T-cell immune response has been established. Some intracellular parasites such as Toxoplasma gondii and Trypanosoma cruzi can establish infection in a variety of cell types, both phagocytic and non-phagocytic. For this purpose they have developed mechanisms actively to invade their host cells. Leishmania, on the other hand, does not seem to contribute actively to the invasion process but rather relies on the phagocytic activity of the macrophage to gain entry. Phagocytosis comprises two linked events: attachment and internalization. It has been known for a long time that promastigote binding and phagocytosis are receptor-mediated events (Chang and Dwyer, 1978; Alexander and Russell, 1992; Mauel, 1996). Initial studies assumed that uptake was carried out by the classical ‘zipper’-type phagocytosis. According to this mechanism, the initial attachment of the microbe to receptors on the phagocyte triggers the recruitment of additional receptors from the surrounding membrane with a concurrent rearrangement of the cytoskeleton. This enables the extension of a pseudopod, which advances along the particle like a zipper engulfing it into a phagosome (Rittig et al., 1998). Recently, a process termed ‘coiling phagocytosis’, which involves asymmetrical occurrence of pseudopodia coils and other multilayered pseudopod stacks, has been suggested as an additional mechanism for parasite uptake (Rittig et a/., 1998). In both processes the complement receptors CRl and CR3 play major roles and may act cooperatively to amplify the effect (Rosenthal et al., 1996). However, uptake by coiling phagocytosis may target the organisms to a cytoplasmic compartment and alter their survival capability (Bogdan and Rollinghoff, 1999). The best characterized interaction of the parasite with the macrophage involves the complement receptors. Leishmania can bind to the complement receptors in three different ways: in the presence of serum by activating C3 directly and binding through C3bi to CR3, through the direct serumindependent binding of the surface protease gp63 to the CR3, and finally via direct binding of lipophosphoglycan to the lectin-like site on CR3 and to CRl (Alexander and Russell, 1992; Mauel, 1996). Engagement of the complement receptors does not trigger the respiratory burst (Wright and Silverstein, 1983) and indeed opsonization by complement increases the survival of L. major in macrophages (Mosser and Edelson, 1987; Mosser and Brittingham, 1997). In humans, the major mechanism for invasion appears to be through the engagement of CR1 on erythrocytes and the classical complement pathway (Dominguez and Torano, 1999). This process does not seem to be present in animal models of disease, in the natural animal reservoir. Macrophage receptors other than CR3/CR 1 have also been implicated in the initial attachment of the parasite, including complement receptor CR4,


9

as well as receptors for fibronectin, mannose receptor and advanced glycosylation end products (Alexander and Russell, 1992). With the availability of ‘knock-out’ mice lacking FcyR, the scavenger receptor and the complement receptor, it should be possible to re-evaluate the contribution of individual host receptors to parasite invasion for each Leishmania species. However, it is likely that, in vivo, multiple receptorligand interactions occur simultaneously depending on the activation state of the macrophage and its microenvironment. 2.1.3. Promastigote Ligands for Host Macrophages The two major promastigote surface molecules, the major protease gp63 or leishmanolysin and the phosphoglycans, are also ligands for attachment to macrophages (Alexander and Russell, 1992). This is the case for both the serum-dependent and the direct binding. L. major gp63 is a zinc metaloprotease, which is abundant on the surface of promastigotes. It is a glycoprotein with unusual N-glycosylation, which suggests that it may be a potential target for the development of a parasitespecific drug (Olafson et al., 1990). The protein is encoded by a family of seven genes. Six of these genes are constitutively expressed in promastigotes. The expression of the seventh gene is developmentally regulated and the protein is produced exclusively in infective metacyclic promastigotes and amastigotes (Joshi et al., 1993). The genes encoding gp63 are situated in a cluster that is present on a similar size chromosome in all species examined (Button et al., 1989). Although very similar in general terms, the gene organization and expression of gp63 in other Leishmania species is somewhat different (Ramamoorthy et al., 1992, 1995; Roberts et al., 1995). Northern blot analysis from both promastigotes and amastigotes revealed a 3-kilobase (kb) mRNA indicating expression in both parasite life stages (Frommel et al., 1989; Medina-Acosta et al., 1989, 1993). Biosynthesis of gp63 is complex, and involves a cleavable N-terminal signal sequence which guides it across the endoplasmic reticulum membrane, and a C terminal hydrophobic signal sequence that is cleaved 25 amino acids from the carboxyl terminus and is replaced by a glycosylphosphatidylinositol (GPI) anchor (Voth et al., 1998). The biological significance of this complex series of events is not yet understood. Surface proteases that are homologous to gp63 have been described for other trypanosomatids including Crithidia which is a parasite of insects (Bouvier et al., 1987; Russell et al., 1991; El-Sayed and Donelson, 1997). The presence of gp63 in these organisms indicates that the protein predates the divergence of Leishmania to become pathogens in vertebrate hosts and suggests an ancestral role for the protein in the insect.

10

E. HANDMAN

Recently, the three-dimensional structure of gp63 has been obtained. It reveals three domains, two of which have folds that have not been previously described for similar enzymes, suggesting another opportunity to exploit gp63 as an antiparasite drug target (Schlagenhauf et al., 1998). The literature describing the mechanism by which gp63 binds to macrophages and that describing the macrophage receptors involved in the process is confusing. Much of the existing literature is summarized elegantly in Mosser and Brittingham (1997). Early data indicated that CR3 was the main receptor for gp63, binding via iC3b as well as directly via the sequence SRYD, in a manner similar to the binding of integrins to the sequence RGDS. More recent studies have suggested that fibronectin receptor rather than CR3 may be the main receptor for SRYD on gp63. With new insights into the biology of complement receptors and the availability of several of the gp63 genes and gp63 gene knock-out parasites, it may be timely to re-examine these issues using site directed mutagenesis and binding assays. 2.1.4. Phosphoglycans The Leishmania phosphoglycan family of molecules comprises glycolipids and glycoproteins containing repeating units of Gal(/31-4)Man(a 1-)PO4 with or without additional glycan side chains (Mengeling et al., 1997; Haynes, 1998). The family includes lipophosphoglycan (LPG), phosphoglycan (PG) as well as the proteins secreted acid phosphatase and proteophosphoglycan (PPG) (Figure 3). 2.1.5. Lipophosphoglycan Lipophosphoglycan is a complex glycolipid present on the surface of all Leishmania promastigotes examined to date. It forms a dense glycocalyx over the entire surface including the flagellum. The history of LPG is instructive in terms of the unexpected and tortuous path of scientific discovery. Studies were originally undertaken by Schnur to explain the observation that promastigotes grown in the presence of immune serum agglutinate and are morphologically abnormal (Adler and Theordor, 1926; Noguchi, 1926; Schnur et al., 1972). From these studies it became apparent that each Leishmania species produces and secretes into the medium a serologically distinct ‘excreted factor’ (Schnur et al., 1972). Excreted factor was said to be ‘haptenic in nature’, sharing antigenic determinants with the whole parasites from which it was derived (Schnur and El-On, 1974; El-On et al., 1979). Serological analysis of excreted factor allowed the identification and classification of isolates of Leishmania based on intrinsic characteristics

11


Membrane

I cap HGal - Man -@H Glycan core H GPI anchor

a LPG

rl

1 cap H G a i - Man -81

PG

rl Protein

---(Gal

- Man -81

EthN

4 GPI anchor

( Serine )-

Protein

44

GPI- anchored PPG Secreted PPG SAP

Figure 3 Schematic representation of the Leishmania phosphoglycans, the GPIanchored lipophosphoglycan (LPG), the related water-soluble phosphoglycan (PG), which lacks the anchor and the glycan core present in LPG, the GPI-anchored proteophosphoglycan (PPG) and its related, secreted PPG. Another proteophosphoglycan is the secreted acid phosphatase (SAP). Abbreviations: EthN, ethanolamine; Gal, galactose; Man, mannose; GPI, glycosylphosphatidylinositol. (Adapted from Beverley S.M. and Turco S., 1998.)

of the organisms rather than on the clinical manifestations of the patient from which they were isolated (Schnur et al., 1973, 1981; Schnur, 1982a). Turco et al. (1984) characterized an acidic glycoconjugate in L. donovani, which behaved like excreted factor. Our laboratory was the first to describe its presence on the promastigote membrane and demonstrate its amphipathic properties (Handman et al., 1984). These properties were subsequently shown to be due to the presence of a covalently attached lipid anchor, which was absent from the soluble form (McConville et al., 1987; McConville and Ferguson, 1993). Early data suggesting the presence of sulphate in LPG (Handman et al., 1984) have not been confirmed. The molecule has no sulphate. This molecule, initially called lipopolysaccharide because of its superficial similarity to bacterial lipopolysaccharide, eventually became known as lipophosphoglycan (Handman et al., 1984; Turco et al., 1984; Handman and Coding, 1985). With the advent of monoclonal antibodies to parasite surface antigens, functional studies examining their role in invasion became possible (Handman and Hocking, 1982; Greenblatt et al., 1983). Monoclonal antibodies to LPG inhibited attachment of the parasite to macrophages, and it was shown that the L. major LPG is one of the parasite ligands

12

E. HANDMAN

interacting specifically with the mammalian macrophage (Handman and Goding, 1985). Subsequent studies on L. donovani confirmed these observations and extended the functional characterization of LPG by showing that it is also a major ligand for the sandfly gut epithelium (Turco, 1988b; McNeely et al., 1990; McNeely and Turco, 1990; Sacks et al., 1994; Beverley and Turco, 1998). Structural analysis revealed that LPG contains four basic domains conserved in all species examined: a 1-0-alkyl-2-lyso-phosphatidylinositol lipid anchor, a glycan core, and a backbone made of repeating phosphodiester linked disaccharide units of Gal(P1-4)Man(a 1-)-PO4 terminating in a neutral oligosaccharide cap (McConville et al., 1990; Turco and Descoteaux, 1992) (Figure 3). The cap structure varies significantly among species, as do the presence or absence of oligosaccharide side chains on the backbone repeat. L. tropica LPG is the most complex, with more than 19 different glycan side chains (McConville et al., 1995). This is followed in complexity by L. major, while the simplest of the cutaneous organisms is L. mexicana with a single 0-linked glucose residue (Ilg et al., 1992; McConville and Ferguson, 1993). The simplest of all the LPGs is that of L. donovani, which contains only the disaccharide backbone and has no side chains (Turco et a[., 1987). In addition to the interspecies and intraspecies polymorphism of LPG, the structure of the molecule is developmentally regulated during the life cycle of the parasite. As will be described in Section 3.3, this is particularly striking in L. major, where LPG undergoes major structural changes in the transition from the immature procyclic to the infective metacyclic promastigotes. The side-chain structure changes, with reduced numbers of galactose residues and an increased number of terminating arabinose residues. Moreover, the number of disaccharide repeating units almost doubles (McConville et al., 1992). This elongation of LPG seems to be important in protection against complement lysis of metacyclic promastigotes. The structure of LPG detected on promastigotes grown in culture is to some extent dependent on the medium used and the culture conditions. The culture conditions also affect the ability of the promastigotes to fulfil the criteria defining metacyclic parasites, such as agglutination by peanut agglutinin (E. Handman, unpublished observations). In the L. major amastigote, LPG is about a 1000-fold less abundant; it becomes a larger but more sparsely substituted molecule (Glaser et al., 1991; Turco and Sacks, 1991; Moody et al., 1993). Some of the side chains are longer containing as many as 10-12 galactose residues (Moody et al., 1991, 1993; Turco and Sacks, 1991). There is a significant heterogeneity in the level of expression of LPG in amastigotes of different species of Leishmania; L. donovani and L. mexicana amastigotes do not seem to express it at all (McConville and Blackwell, 1991; Bahr et al., 1993).

CELL BIOLOGY OF LNSHMANlA

13

LPG has a distinct GPI anchor structure compared to the Leishmania protein GPIs, and these are in turn distinct from mammalian protein GPIs (McConville and Ferguson, 1993). Another unusual feature of the Leishmania LPG molecule is the presence of a galactofuranose residue in the glycan core. This sugar is not present in mammalian glycoconjugates. The biological significance of the GPI anchors and the presence of the galactofuranose is not clear, but their uniqueness makes them potential targets for specific drug design. LPG is a virulence factor essential for parasite survival in both the insect vector and mammalian macrophage. In the case of L. major promastigotes, studies by Handman and Goding (1 985) and subsequent work identified the galactose-containing side chains Gal( /31-3)Gal( /31-3)Ga1(/31-3)Gal(/314)Man(al-)P04 of LPG as the domain involved in the interaction with host cells (Kelleher et a[., 1992). Amastigotes also use LPG to bind to macrophages but in this case the epitope includes the disaccharide repeats of the backbone, which is more accessible in the sparsely substituted amastigote LPG (Kelleher et al., 1993, 1995). As originally described by Handman and colleagues (Handman et a[., 1984; Handman and Goding, 1985), the glycoconjugate exists in two forms: a membrane-bound amphipathic form and a hydrophilic form found in parasite culture supernatant. The structural differences between these forms has now been established. The water-soluble fragment of LPG is known as phosphoglycan (PG), and is released into the culture medium by promastigotes (Figure 3 ) . PG consists of the LPG repeating units and the terminating cap structure, but does not have hydrophobic properties and lacks a GPI anchor (Handman et al., 1984; Greis et al., 1992; Ilg et al., 1994). However, it is still not known whether PG is a hydrolysis product of LPG, or whether it is synthesized separately and secreted from the flagellar pocket. Nor is it known whether PG is also produced in vivo in the sandfly. While it is quite clear that PG is one of the main components of the original excreted factor described above, it may not be the only one. With the discovery that glycan chains are shared by LPG and several proteophosphoglycans, much of the literature will have to be revisited (see below). No function has been proposed for PG, but LPG glycans have been shown to appear on the surface of macrophages soon after invasion by promastigotes (Handman, 1990; Tolson et al., 1990). Some of these molecules are the hydrophobic form of LPG, and others are hydrophilic and may be PG. Studies on L. donovani LPG have demonstrated that it is also involved in macrophage binding and it acts as a virulence factor (reviewed by Turco and Descoteaux, 1992: Beverley and Turco, 1998). The critical role of LPG in virulence for the mammalian host is supported by the fact that L. major and L. donovani mutants lacking LPG are avirulent (Elhay et al., 1990; McNeely

14

E. HANDMAN

and Turco, 1990; Opat et al., 1996). Moreover, intercalation of LPG into the surface membrane of the mutant organisms restored their virulence to some extent (Handman et al., 1986; McNeely and Turco, 1990). More recently, using elegant genetic approaches, mutant genes from several LPG-null mutants have been identified and cloned (Ryan et al., 1993; Beverley and Turco, 1995, 1998). Wild-type genes were capable of restoring the production of LPG. In some of the mutants, transfection with the wildtype gene restored virulence, thus fulfilling Koch’s postulates (Beverley and Turco, 1998). The functions of these genes have not been fully elucidated. Based on their overall structure, some may be glycosyltransferases, although this awaits definitive biochemical characterization. In addition to its role in attachment to macrophages and invasion, LPG has been shown to have many immunomodulatory activities. It scavenges hydroxyl radicals and superoxide ions which are normally released upon activation of NADPH oxidase by phagocytosis (Mauel, 1996; Bogdan and Rollinghoff, 1998). Intact L. donovani promastigotes have been shown to block protein kinase C activity, and purified promastigote LPG has also been shown to have this activity (McNeely and Turco, 1987; McNeely et al., 1989; Descoteaux and Turco, 1993). 2.1.6. Proteophosphoglycans While the first phosphoglycans to be described were the glycolipid LPG and the polysaccharide PG, a family of phosphoglycan-modified proteins has now been added to the list (Mengeling et al., 1997). To date, three protein members of this family have been characterized. These are the secreted acid phosphatase (SAP) from L. mexicana, a high molecular weight filamentous PPG secreted by promastigotes of L. major (pPPG), and a smaller and structurally distinct PPG secreted by amastigotes of L. mexicana (aPPG). SAP is secreted from the flagellar pocket, the specialized site for secretion and endocytosis (Stierhof et al., 1994). The enzyme is monomeric or oligomeric in structure depending on the organism (Ilg et al., 1991; Stierhof et a[., 1994). In L. mexicana the enzyme is encoded by two genes, and both products are enzymatically active. SAP is a serine- and threonine-rich molecule, and many of its serine residues have a novel type of modification, phosphoglycosylation, the role of which is not yet known (Haynes, 1998). Many of the glycans present on SAP are shared with LPG. SAP is present in most Leishmania species but its biological function is still not understood. SAP is not present in L. major, and ablation of the L. mexicana SAP genes has no effect on parasite growth in vitro or virulence in vivo (Wiese, 1998). Promastigotes of many Leishmania secrete a filamentous proteophosphoglycan which forms gel-like networks and is seen at the centre of

CELL BIOLOGY OF LElSHMANlA

15

parasite rosettes in vitro (Stierhof et al., 1994; Ilg et al., 1996). pPPG from L. major is a large and highly glycosylated mucin. Reminiscent of vertebrate proteoglycans, pPPG has a predominance of carbohydrate (76%) and only 4% amino acids. About half the amino acids are serine, which, together with alanine and proline, form over 80% of the protein backbone (Ilg et al., 1996). The majority of the serines are phosphoglycosylated with LPG-like phosphodiester-linked PG chains. In a striking parallel to LPG and PG, PPG also is found in two distinct forms, a water-soluble secreted form and a GPI-anchored membrane-bound form (A. Piani et al., unpublished data). A gene encoding the membranebound pPPG has been isolated (Ilg et al., in press). The deduced amino-acid sequence contains a hydrophobic C-terminal domain but no cytoplasmic tail, consistent with GPI anchorage. Adjacent to this region is a nonrepetitive domain, while the main body of the open reading frame consists of a large number of repeats of a basic unit of 12- 15 amino acids rich in serine, alanine and proline. At the amino-terminal region there is a second nonrepetitive domain of about 600 amino acids. While the definitive demonstration of the function of pPPG awaits the characterization of gene knock-out organisms, it is already clear that watersoluble pPPG plays a role in the interaction of the parasite with the sandfly (Y.-D. Stierhof, T. Ilg, Y. Schlein and R.L. Jacobson, unpublished data; see also Section 3.2). Purified pPPG binds to macrophages, is internalized and can be detected in the lysosomal compartment (Piani et al., 1999). PPG can also be detected in amastigotes and in parasite-free vesicles in infected macrophages. In view of the striking structural similarities between LPG and PPG, it will now be important to re-examine their relative contribution to a variety of functions previously attributed to LPG, in particular in the amastigote, which displays much less LPG on its surface. A particularly intriguing question is why evolution has led to the production of two polymers with similar or identical side chains, but with such different backbones (phosphodiester-linked sugars versus amino acids).

2.2. Promastigote to Amastigote Differentiation in the Macrophage

2.2.1. Parasitophorous Vacuole Formation and Microbicidal Mechanisms

The first stage in Leishmania infection involves the uptake of promastigotes into a membrane-bound phagosome, which is contiguous with the outer plasma membrane of the macrophage. At this stage the parasite is still topographically in the extracellular environment. The phagosome then becomes modified by fusion with secondary lysosomes, resulting in the

16

E. HANDMAN

phagolysosome or parasitophorous vacuole (PV) (Chang, 1983; Chang and Fong, 1983). The PV is an acidic compartment, rich in microbicidal peptides and hydrolytic enzymes (Antoine et al., 1998). With the transition from the sandfly to the mammalian host, the promastigotes face two major environmental changes, a temperature shift to 35537°C and a pH shift to around pH 5. The organisms sense this new environment and transform into the obligatory intracellular amastigotes (Antoine et al., 1998) with loss of flagellum, closing off of the flagellar pocket, drastic reduction in size and major changes in gene expression. The details of how this transition is triggered and carried out are unclear, but seem to involve unknown factors in addition to the change in pH and temperature. The availability of the complete Leishmania genome sequence may soon provide some tools to help elucidate this process (Blackwell, 1997; Ivens and Smith, 1997; Foote et al., 1998). The elucidation of the mechanisms by which the transition between promastigote and amastigote is mediated will require much more knowledge than just the sequences of the genes. The transition may be regarded as a process of differentiation, and the key questions are how individual genes are activated and inactivated, how a stable phenotype is maintained, and how the changed environment triggers a genetic reprogramming. Although all PVs containing Leishmania share many features, such as mildly acidic pH and the presence of hydrolases and lysosomal membrane markers such as LAMP-1 and LAMP-2, there are significant differences between the PVs produced by different Leishmania species. For example, L. mexicana and L. amazonensis produce large PVs containing many amastigotes arranged around the periphery and attached to the membrane, while L. major and L. donovani produce small PVs with little space around the amastigotes. Some differences in the PVs may be related to the life-cycle stage of the parasite producing it. For example, the aPPG produced by L. mexicana amastigotes induces the formation of large vacuoles in macrophages in the absence of parasites (Peters et al., 1997). In contrast, pPPG from L. major promastigotes causes only modest vacuolation in macrophages (J.-C. Antoine, personal communication). Although the mechanism involved in the formation of Leishmania PV and the role of the intracellular pathogen in its development are not well understood, there are some similarities with the vacuole formation induced by Helicobacter pylori (Antoine et al., 1998). In the case of H. pylori, the toxin Vac A produces large vacuoles in many cell types by inhibiting phosphatidylinositide 3-kinase, an enzyme required for the fusion of late endosomes (Antoine et al., 1998). Vac A was shown to bind to, and to be internalized by, the target cells (Garner and Cover, 1996; Massari et al., 1998).Vac A has also been shown to have immunomodulatory effects and to interfere with antigen presentation by B cells (Molinari et al., 1998).


17

In contrast to PPG, which, in the case of L. mexicana and L . amazonensis induces large vacuole formation in macrophages, promastigote LPG has been shown transiently to prevent the fusion of phagosomes with lysosomes (Desjardins and Descoteaux, 1997). Whether the transient sojourn of the parasite in this type of the phagosome provides the trigger for the initiation of transformation to amastigotes is not known. Infection by Leishmania seems to alter some but not all the microbicidal processes of the macrophage. The vacuolar pH is maintained, the hydrolases are targeted to the PV normally and vesicular traffic does not seem to be disturbed (Russell et al., 1991, 1992; Russell, 1995; Antoine et al., 1998). On the other hand, infection inhibits the production of superoxide (02) and H202, which is one of the major macrophage microbicidal effector mechanisms (Murray, 1986). In addition, infection of cells by Leishmania seems to induce the rapid and transient production of a subset of chemokines. Among these is the monocyte chemoattractant protein 1, which may be important in attracting into the lesion ‘safe targets’ in the form of immature monocytes. These can be infected but, because they express little MHC class I1 on their surface, they present antigen poorly and do not kill the parasites (Racoosin and Beverley, 1997). 2.2.2. Amastigote Survival in the Macrophage What are the biochemical changes that make the amastigote so well adapted to the hostile intracellular environment of the PV with its acidic pH and abundance of hydrolases? Early work from our laboratory suggested that the membrane proteins of the amastigotes were more resistant to proteolysis compared to promastigote membrane proteins (Handman and Curtis, 1982). The amastigote metabolism is adapted to an acidic pH (Glaser et al., 1988) and amastigotes are thought to exploit the proton gradient across their membrane formed under acidic conditions to drive the active transport of glucose and amino acids (Zilberstein, 1991; Zilberstein and Shapira, 1994). This mechanism may actually contribute to the maintenance of the acidic pH in the PV. Another metabolic change, noted by Janovy (1967), was a drastic reduction in the rate of respiration suggesting a switch to anerobic metabolism. Several amastigote-specific gene products have been identified, such as a 3’-nucleotidase (Bates, 1993), the amastigote-specific protein A2 of L. donovani (Zhang and Matlashevski, 1997) and a mitogen-activated protein (MAP) kinase in L . mexicana (Wiese, 1998). An amastigote-enriched histone H1 gene has also been identified (Fasel et al., 1994). In addition, members of multigene families, such as the parasite surface antigen 2 (PSA-2) or gp63 of L . major are differentially expressed in

18

E. HANDMAN

amastigotes (Handman et al., 1995). The structure of the GPI anchor of the amastigote PSA-2 polypeptide is different from that of the three PSA-2 polypeptides expressed by promastigotes. In contrast to the promastigote forms, the amastigote GPI is resistant to hydrolysis by phosphatidyl inositol-specific phospholipase C from B. thuringiensis (E. Handman, unpublished data). When the amastigote PSA-2 gene is expressed in promastigotes by transfection, the GPI anchor is susceptible to hydrolysis, suggesting a stage-specific control mechanism in anchor biosynthesis (E. Handman, unpublished data). It would seem a reasonable hypothesis that some of these amastigote-specific gene products contribute to the ability of the amastigotes to establish in the macrophage and to evade its microbicidal activity, but at present direct evidence is lacking. An intriguing characteristic of the amastigote surface is the increased ratio of glycolipids to proteins. The endogenous glycolipids and the host-derived sphingolipids incorporated into its membrane represent a significantly larger proportion of the surface components compared to promastigotes (McConville and Ferguson, 1993). Among them, the glycoinositol phospholipids (GIPLs) are the most abundant, forming a densely packed and morphologically distinct protective coat. The GIPLs are structurally related to LPG but distinct from it. They possess a unique GPI anchor, containing lipids which are different from both protein GPIs and LPG (Ralton and McConville, 1998). It has been suggested that they play a role in the regulation of the physicochemical properties of the amastigote membrane by making it more resistant to enzymatic attack in the PV (Ralton and McConville, 1998). In addition, GIPLs have been shown to contribute to amastigote survival in the macrophage by directly inhibiting microbicidal activities such as NO production (Winter et al., 1994; Proudfoot et al., 1995). 2.3. Mechanisms of Parasite Persistence

2.3.1. Invasion of Macrophages and Dendritic Cells by Amastigotes Although the initiation of infection is due to the promastigote, the maintenance of infection in the mammalian host relies on the amastigotes and their ability to replicate in macrophages, and to exit and re-infect new host cells. Much progress has been made in the elucidation of the host and parasite molecules, and of the mechanisms involved in early promastigote attachment and uptake by macrophages. Much less is known about the interaction of the obligatory intracellular amastigote and its host cells in an already established infection.


19

In vitro studies indicate that LPG is a major ligand on L. major amastigotes, but this cannot be the case for Leishmania species that do not express significant amounts of LPG on their surface. For those it will be interesting to explore the role of PPG. In the case of L. amazonensis amastigotes, an undefined heparin-binding molecule has been implicated in the attachment to macrophages (Love et ul., 1993), as have amastigotespecific glycosphingolipids (Straus et al., 1993). On the host side, recent data implicate the Fc receptor for IgG as a major contributor to infection of macrophages by L. mexicana amastigotes in vivo. The complement receptor CR3 and the mannose receptor are also important (Peters eta]., 1995). It has been suggested that the Fc receptor may also play a role for L. major amastigotes. A major pathogenic role for Fc receptor in infection is difficult to reconcile with the very low amount of antibody present in the tissue of L. major-infected individuals and with the massive infection of hypothymic nude mice that lack IgG antibodies. It is likely that, as part of the ‘arms race’ between host and parasite, multiple receptorligand interactions have evolved to allow parasitism. There is now abundant evidence that a host cell carries amastigotes from the initial site of infection in the skin to the draining lymph nodes, where the antigen presentation to the naive T cells occurs and where parasites persist indefinitely (Aebischer et al., 1993; Moll, 1993a,b,c; Moll et al., 1993, 1995a,b). The cell that ferries the amastigotes from the skin to the lymph node appears to be a dendritic cell (Langerhans cell). The receptor on the dendritic cells responsible for the interaction with amastigotes is not known nor is the ligand on the amastigote. Dendritic cells are required to initiate primary T-cell responses (Caux el al., 1995). Macrophages can only present antigen to T cells that have already been primed (Caux et al., 1995). In contrast to the infected macrophages, which seem to be impaired in antigen presentation (see below), the infected dendritic cells are competent to present antigen and initiate T-cell immune responses to the parasite (Moll, 1993~). An intriguing question in leishmaniasis is the homing of the different Leishmania species to different organs. What is the contribution of the parasite and what is the contribution of the host? Are dendritic cells involved in this migration? How early in infection does it occur? 2.3.2. Evasion and Subversion of the Host Immune Response Leishmaniasis is a chronic disease; the infection is slow in turning on the host protective macrophage-activating immune responses. The parasites have evolved numerous ways to interfere with the host immune responses, including modulating cytokine production, inhibiting antigen presentation

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and turning off co-stimulatory molecules necessary for activation of antigenspecific T cells. L. mexicana, L. major and L. braziliensis trigger the production of transforming growth factor /3 (TGF-/3) and interleukin 10 (IL-lo), which inhibit killing of the intracellular organisms (Bogdan and Rollinghoff, 1998, 1999). Recovery from infection has been shown to be critically dependent on the macrophage-activating cytokine IL-12 (Reiner and Locksley, 1995), and infection with promastigotes inhibits production of IL-12 and TNF-a (Reiner et al., 1994). Some of the parasite molecules involved in modulating the immune response of the host have been identified but the mechanisms by which they act on the macrophage biology are mostly not understood. For example, LPG and PPG suppress IL-1 and TNF-a production in response to bacterial LPS. LeIF, a Leishmania homologue of the initiation factor 4A has been shown to modulate IL-12, IL-10 and TNF-a expression in monocytederived antigen presenting cells (Probst et al., 1997). Parasite-driven mechanisms operating at the level of the PV also have direct effects on the recognition of the parasite by T cells. The parasite causes a reduction of host MHC class I1 molecules available for binding to parasite antigens (Handman et al., 1979; Reiner et al., 1987; Antoine et ul., 1991; Lang et al., 1994a,b), which presumably helps prevent its detection in the macrophage by T cells. Interestingly, in the case of L. amazonensis and to some extent L. major, this seems to be achieved by selective and active sequestration and degradation of class I1 antigens by the amastigotes (Antoine et al., 1998). In L. amazonensis, the MHC class I1 molecules seem to accumulate in amastigote-specific organelles known as megasomes, and it is possible that amastigote-specific proteases are involved in the hydrolysis of the MHC molecules. An additional mechanism which reduces antigen presentation by infected macrophages has been described in L. mexicana. The availability of parasitederived antigens for presentation to the immune system seems to be restricted to macrophages containing dead organisms (Wolfram et al., 1995, 1996). This might imply that the death of the parasite allows the release of parasite antigens which then find their way to the macrophage surface. More interestingly, it could suggest the existence of active suppressive mechanisms that require the parasite to be alive. These immune evasion mechanisms mean that many, if not all, infected macrophages remain immunologically silent. This could provide an explanation for the slow development of the host protective effector mechanisms and possibly for the long-term persistence of the parasite in the immune individual (Aebischer, 1994; Bogdan et al., 1996). The mechanisms allowing indefinite persistence of the parasite in the presence of an otherwise host protective immune response are still poorly


21

understood (Bogdan et al., 1996). One effect of the parasite on the macrophage described recently is inhibition of apoptosis through induction of ‘pro-survival’ cytokines such as macrophage colony-stimulating factor (M-CSF), tumour necrosis factor a (TNF-a) and IL-6 (Moore and Matlashewski, 1994; Moore et al., 1994; Antoine et al., 1998). This phenomenon may be responsible for the persistence of amastigotes by extending the life of the host cell. It is also possible that the persistent organisms reside in cells other than macrophages, for example, dendritic cells or fibroblasts (Bogdan et al., 1996).

3. THE INTERACTION OF LElSHMANlA WITH THE SANDFLY 3.1. Parasite Differentiation in the Blood Meal

When feeding on blood as opposed to fruit, the female sandfly is a pool feeder that uses its mandibles and maxillae to cut a wound in the host skin, and sucks up the blood that accumulates. Infected macrophages are taken up from that pool. The blood meal becomes enclosed in a sac-like peritrophic membrane, which is secreted by the midgut epithelium and consists of chitin embedded in a protein-carbohydrate matrix (KillickKendrick, 1990a,b). Amastigotes continue to undergo a few cell divisions in the blood meal but the new environment is sensed by the organisms, and metabolic changes are set in train leading to the transformation of the non-motile, aflagellar amastigote into the motile flagellated promastigote (Killick-Kendrick, 1990a). Just as the amastigote has evolved mechanisms to survive in the mammalian macrophage, so the promastigote has evolved mechanisms to promote life in the insect host. The nomenclature used for the different developmental stages of Leishmania in the sandfly has been adopted from the nomenclature of the African trypanosomes, and includes the procyclic or midgut form and the metacyclic or mature infective form (Vickerman and Preston, 1976; Sacks, 1988). The early procyclic promastigotes are eliptical in shape, measure only 6-8 pm in length, divide rapidly and, after a few days, escape from the disintegrating peritrophic membrane and migrate forward to the thoracic midgut and the cardiac valve (Schlein, 1993). Procyclic promastigotes continue to divide and attach to the microvilli of the midgut, particularly in the thoracic section and the cardiac valve. Later, some invade the oesophagus and the pharynx where they attach to the cuticle lining with their flagella, which form plaques called hemidesmosomes (Warburg et al., 1989; Lang et al., 1991; Schlein, 1993). Surprisingly, it seems that the flagella

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may even penetrate into the epithelial cells, presumably increasing the strength of the parasite anchorage (Walters et al., 1987). A parasite molecule present along the flagellum and at its tip has been implicated in this specific interaction (Warburg et al., 1989). Further differentiation and maturation of the promastigote population occurs from day 5-8 onwards, and is reflected by major changes in morphology and biochemistry. At this stage, the metacyclic form of the promastigote becomes dominant in the population (Sacks and Perkins, 1984; Sacks, 1989). The metacyclic promastigotes are slender, highly motile organisms with small bodies and long flagella. The metacyclic promastigotes are now ready to be delivered by the sandfly to the mammalian host at the next blood meal.

3.2. Establishment of Infection in the Sandfly

The sandfly is not just a vector for delivery of organisms to mammalian hosts. It is itself a host and, as such, the interaction of the parasite with the sandfly is just as complex as its interaction with the mammalian host. The parasites undergo a series of developmental modifications which on the one hand allow them to survive in the gut environment and on the other hand make it possible for transmission to the mammalian host. There is evidence for significant specificity in the interaction of the parasite with the sandfly. Certain species of Leishmania can be transmitted only by particular species of Phlebotomus. What determines this hostparasite specificity? Studies dissecting the genetics of P. papatasi susceptibility to a single isolate of L. major showed that individual flies varied in susceptibility (Wu and Tesh, 1990b) and that this variation was due to multiple genes (Wu and Tesh, 1990a). Species-specific differences in vectorial competence have been correlated with the ability of the parasites to establish in the gut. The parasites have to withstand the effects of the proteases secreted by the fly in response to the blood meal. They also have to anchor themselves in the peristaltic environment of the gut to prevent expulsion through the anus. LPG and the related water-soluble polysaccharide PG are thought to protect the parasites from the gut hydrolytic environment (Schlein, 1993; Pimenta et al., 1994). There seems to be some specificity in the ability of these molecules to protect the parasites. L . major but not L. donovani PG was able to inhibit the proteolytic activity of the gut enzymes, and to increase survival of the L. major but not L . donovani parasites in P. papatasi, the natural host of L. major. This sandfly is not a host for L. donovani (Schlein and Romano, 1986; Schlein, 1993).


23

One of the parasite molecules implicated in the anchorage to the gut is the LPG coat, which forms a thick glycocalyx on the promastigote surface. The receptor on the gut epithelium of P. papatasi, which is specific for L. major, appears to recognize galactose residues on the side chains of promastigote LPG. Side chains are absent from L. donovani LPG, which consists of only the backbone Gal(,B1-4)Man(a l-)PO4 terminating with a neutral cap. The structural differences in LPG may explain the inability of L. donovani to colonize this particular sandfly species. The importance of the galactosecontaining side chains for the establishment of L. major infection in this particular sandfly has been unequivocally demonstrated by the use of a L. major mutant lacking the gene for the ,Bl,3-galactosyltransferase(Butcher et af., 1996). This mutant, which produces an LPG similar to L. donovani devoid of side chains, cannot bind to the midgut and cannot sustain a successful infection in P. papatasi. As described earlier, the L . major promastigotes display a surface-bound PPG, which is decorated with carbohydrate side chains similar in structure to those present on LPG (Ilg et al., 1996; A. Piani et al., unpublished data). This molecule may also contribute to the binding to gut epithelium. The next stage of parasite development is accompanied by the escape from the peritrophic membrane. The haemoglobin in the blood meal is digested, possibly by the parasite major surface protease gp63 (Schlein, 1993), following which the membrane is lysed by parasite chitinases (Schlein et al., 1991; Schlein, 1993). The cues for the parasites’ migration to the thoracic midgut and cardiac valve and for their subsequent differentiation remain to be elucidated. Turco (1988a) has made the interesting suggestion that sugar meals that are taken into the sandfly crop and are delivered into the gut may facilitate the parasite migration by competing for binding to the receptors for LPG on the epithelial cells. However, parasites can complete their differentiation even in the absence of sugar meals (Schlein, 1993). Certain bacteria possess a sophisticated signalling strategy called ‘quorum sensing’ in which the density of the population is sensed and virulence genes are expressed when the population reaches a certain size. The quorum sensing signal is triggered by the concentration of a secreted microbial molecule (Straws and Falkow, 1997). It would be interesting to investigate the existence of quorum sensing as a mechanism for the changes in the parasite developmental programme in the gut.

3.3. Metacyclogenesis

The parasite morphotypes present in the thoracic midgut and proboscis have always been considered strong candidates for the initiation of infection because of their proximity to the wound (Adler, 1964). The presence of

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different morphotypes along the digestive tract of the fly during the differentiation of the parasites from the amastigote to the promastigote form also suggested potential differences in the ability of individual forms to infect the mammalian host. Sacks and Perkins (1984) provided the definitive demonstration that the mature or metacyclic non-dividing promastigotes present in the midgut are the infective stage of the organism by showing that promastigotes taken from sandflies 3 days after infection were avirulent, whereas those taken after 7-8 days were infective (Sacks and da Silva, 1987). Subsequently, they showed that metacyclic promastigotes are also present in in vitro cultures in a stationary phase of growth but not in the logarithmic phase (Sacks, 1989). The small population (2- 10%) of metacyclic organisms can be isolated from these cultures by negative selection for agglutination by the galactose-binding lectin peanut agglutinin (PNA; Sacks, 1989). Procyclic or immature promastigotes bind PNA and are agglutinated by the lectin, whereas the metacyclic promastigotes remain in suspension. It should be noted, however, that the parasites that are not agglutinated by PNA still bind the lectin, and the precise basis for the agglutination by PNA remains to be determined. Metacyclogenesis is accompanied by ultrastructural and biochemical changes in the parasite, in particular at the cell surface. One of the most significant changes is thickening of the glycocalyx owing to modifications in the structure of LPG (Pimenta et al., 1989, 1991, 1992, 1994). Changes in glycosylation of LPG lead to increases in the length of its backbone and in the masking of galactose residues on its side chains, the targets for PNA binding, by arabinose (McConville et al., 1992; McConville and Ferguson, 1993). A second surface molecule whose expression is upregulated during metacyclogenesis is the major promastigote surface protease, gp63 (Sacks and Perkins, 1984; Russell and Wright, 1988; Kweider et al., 1989). Not only is gp63 more abundant but in L. braziliensis there seems to be a developmentally regulated isoform expressed specifically in metacyclic promastigotes (Kweider et al., 1987) correlating with their increased virulence. Although most of the work on metacyclogenesis has been done in L. major, metacyclic forms have also been observed in L. donovani (Howard et al., 1987) and L. panamensis (Walters et al., 1989a,b). Since these parasites lack the galactose-containing side chains that bind PNA and define metacyclic forms, the molecular basis for the classification of these forms as metacyclic is not clear.

3.4. Transmission to the Mammalian Host

Infection with Leishmania causes havoc in the sandfly, most obviously to its ability to feed. Infected flies seem to probe multiple times until successful


25

feeding is achieved. It may be significant that each of these ‘unproductive’ bites is infective (Killick-Kendrick, 1979; Jefferies et al., 1986; Warburg and Schlein, 1986). A hypothesis has been put forward by Killick-Kendrick (1979) that multiple probing is due to the blockage of the fly midgut, cardia and stomodeal valve by a plug of carbohydrate-rich gel-like material. There is now convincing evidence that this plug is formed by the secreted promastigote PPG, which forms large filaments containing masses of promastigotes (Y.-D. Stierhof, T. Ilg, E. Handman and Y. Schlein, unpublished data). Metacyclic promastigotes appear mainly anterior to this plug and their motility is unimpeded by the gel (Lawyer et al., 1990). Backflow from the plug into the wound during unsuccessful feeding would carry the parasites into the wound. An important factor in the mechanics of transmissibility of the parasites by the fly is the damage to the cardiac valve induced by parasite-derived molecules (Schlein, 1993). In infected flies the damaged valve remains open and the suction of the food pump occurs in both directions, with the gut content including parasites, being drawn back, thus mixing with the newly drawn blood meal. As the pump contracts, the mixture is released in both directions. 3.4.1. The Role of Sandfly Saliva in Promastigote Virulence During the feeding process, sandflies inject saliva into the skin of the mammalian host, as do many blood-sucking insects. The saliva facilitates the location of suitable blood vessels and prevents blood clotting (Lehane, 1991). In addition, the sandfly saliva contains potent vasodilating compounds which presumably increase blood flow to the bite (Ribeiro et al., 1989). The sandfly saliva also acts as a virulence factor for the Leishmania promastigote. It facilitates infection by increasing both ‘the lesion size and the parasite survival (Titus and Ribeiro, 1988). Parasite survival is critical to the establishment of infection because, unlike the situation in the laboratory where large numbers of organisms are injected into the host, in nature, sandflies introduce only 10- 100 organisms at each bite (Warburg and Schlein, 1986). Insect saliva does not act directly on the parasite, but rather through the modulation of macrophage-killing mechanisms, possibly via inhibition of the ability of interferon (IFN-y) to activate macrophages to produce NO, one of the major effector molecules responsible for parasite killing (Hall and Titus, 1995). Recent data indicate that the mechanism of downregulation of NO production may involve inhibition of protein phosphatase activity, blocking signal transduction pathways (Waitumbi and Warburg, 1998). The importance of IFN-y and NO in the early stage of infection is not clear but the effect of the saliva is

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surprisingly long lasting. The components of saliva that mediate these effects on macrophages have not been isolated, but one appears to be low molecular weight, soluble in ethanol and resistant to proteases, DNAse and RNAse. Another important bioactive component in sandfly saliva is the vasodilator and erythema-inducing peptide maxadilan (Lerner and Shoemaker, 1992). Studies by Warburg et al. (1994) indicate that maxadilan may influence the clinical manifestations of disease, possibly by modifying the spreading of the parasites from the skin to the viscera.

4. CONCLUDING REMARKS 4.1. Cell Biology in the Post-genome Era

At the time of writing, at least 13 bacterial genomes have been completely sequenced, 49 are currently in progress in the public domain and several others are held in private companies (Saunders and Moxon, 1998). For more complex organisms such as protozoa, the pace has been slower. The Plasmodium fakiparum genome project is the most advanced, but other parasites such as the African Trypanosomes and Leishmania are on the way (Blackwell, 1997). As a prelude to the genome project, a karyotype map for L. infantum and L. major have been prepared (Wincker et al., 1996) and a physical map has been developed by Ivens and Smith (Blackwell, 1997). Mapping studies have suggested that the overall chromosomal structure and gene order have been maintained in all Old World Leishmania species and therefore a single strain of L. major was chosen for sequencing (Blackwell, 1997). What would the complete sequence of the Leishmania mean to the cell biologist? We will have the sequence of every enzyme and structural protein, all the virulence factors and all the potential drug targets (Foote et al., 1998). However, the availability of the sequence will not translate immediately into function. How should we go about exploiting the knowledge of the Leishmania genome? How can we identify the function of important genes, find virulence determinants, stage-specific genes and specific drug targets? Homology to known genes and analysis of clusters of orthologous groups of genes are obvious approaches, but some genes may be missed because of evolutionary divergence in sequence or because specialized genes may have evolved to exploit the intracellular environment of the parasitophorus vacuole or the sandfly gut (Strauss and Falkow, 1997; Saunders and Moxon, 1998). From the completed sequence of bacteria, yeast and the nematode C . elegans, it is becoming clear that a significant proportion of open reading


27

frames have no identifiable homologues or known function. Characterization of these sequences will require ingenuity, luck and novel approaches. Gene knock-out is relatively straightforward in Leishmania. Recently, the value of transposon mutagenesis for identification of gene function has been demonstrated in Leishmania (Gueiros-Filho and Beverley, 1997). Modifications to the in vivo expression technology, pioneered in the Sabnonella system, may facilitate the study of differential gene expression in the Leishmania amastigote (Saunders and Moxon, 1998). Finally, the development of the chip microarray technology promises to revolutionize the way we think and approach gene expression and function, in particular the coordinated expression of multiple genes. At present, microchip technology lacks the sensitivity to detect transcripts that are of low abundance but may nonetheless be very important. The last decade or so in molecular parasitology has been dedicated to the reduction of complex systems to individual components. The next decade should see the complete sequence of many genomes, including the Leishmania genome. The challenge will be to identify the function of these genes, to put the pieces together, and to make sense of the total organism.

ACKNOWLEDGEMENTS The work from the investigator’s laboratory has been supported by the Australian Health and Medical Research Council and the UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases. I am particularly grateful to my long-standing collaborators Jim Goding and Tony Bacic for helpful discussions and critical input.

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Voth, B.R., Kelly, B.L., Joshi, P.B., Ivens, A.C. and McMaster, W.R. (1998). Differentially expressed Leishmania major gp63 genes encode cell surface leishmanolysin with distinct signals for glycosylphosphatidylinositol attachment. Molecular and Biochemical Parasitology 93, 3 1-41. Waitumbi, J. and Warburg, A. (1998). Phlebotomus papatasi saliva inhibits protein phosphatase activity and nitric oxide production by murine macrophages. Infection and Immunity 66, 1534- 1537. Walters, L.L., Modi, G.B., Tesh, R.B. and Burrage, T. (1987). Host-parasite relationship of Leishmania mexicana mexicana and Lutzomyia abonnenci (Diptera: Psychodidae). American Journal of Tropical Medicine and Hygiene 36, 294-3 14. Walters, L.L., Chaplin, G.L., Modi, G.B. and Tesh, R.B. (1989a). Ultrastructural biology of Leishmania ( Viannia) panamensis ( = Leishmania braziliensis panamensis) in Lutzomyia gomezi (Diptera: Psychodidae): a natural host-parasite association. American Journal of Tropical Medicine and Hygiene 40, 19-39. Walters, L.L., Modi, G.B., Chaplin, G.L. and Tesh, R.B. (1989b). Ultrastructural development of Leishmania chagasi in its vector, Lutzomyia longipalpis (Diptera: Psychodidae). American Journal of Tropical Medicine and Hygiene 41, 295-3 17. Warburg, A. and Schlein, Y. (1986). The effect of post-bloodmeal nutrition of Phlebotomus papatasi on the transmission of Leishmania major. American Journal of Tropical Medicine and Hygiene 35, 926-930. Warburg, A,, Saraiva, E., Lanzaro, G.C., Titus, R.G. and Neva, F. (1994). Saliva of Lutzomyia longipalpis sibling species differs in its composition and capacity to enhance leishmaniasis. Philosophical Transactions of the Royal Society of London 345, 223-230. Warburg, A., Tesh, R.B. and McMahon, P.D. (1989). Studies on the attachment of Leishmania flagella to sand fly midgut epithelium. Journal of Protozoology 36, 613-617. Wiese, M. (1998). A mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host. EMBO Journal 17, 2619-2628. Wincker, P., Ravel, C., Blaineau, C., Pages, M., Jauffret, Y . , Dedet, J.-P. and Bastien, P. (1996). The Leishmania genome comprises 36 chromosomes conserved across widely divergent human pathogenic species. Nucleic Acids Research 24, 1688-1694. Winter, G., Fuchs, M., McConville, M.J., Stierhof, Y.-D. and Overath, P. (1994). Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid. Journal of Cell Sciences 107, 2471-2482. Wolfram, M., Ilg, T., Mottram, J.C. and Overath, P. (1995). Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites. European Journal of Immunology 25, 1094- 1100. Wolfram, M., Fuchs, M., Wiese, M., Stierhof, Y.-D. and Overath, P. (1996). Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface. European Journal of Immunology 26, 31533162. World Health Organization (1995). Report on the Consultative Meeting on Leishmania/HIV Co-infections, September, 1994, pp. 6-7. Rome: Istituto Superiore di Sanita/Geneva: World Health Organization.


39

World Health Organization (1998). Advances in battle against leishmaniasis. T D R News 57. Wright, S.D. and Silverstein, S.C. (1983). Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from human phagocytes. Journal of E.xperimenta1 Medicine 158, 2016-2023. Wu, W.K. and Tesh, R.B. (1990a). Genetic factors controlling susceptibility to Leishmania major infection in the sand fly Phlebotomus papatasi (Diptera: Psychodidae). American Journal of Tropical Medicine and Hygiene 42, 329-334. Wu, W.K. and Tesh, R.B. (1990b). Selection of Phlebotomus papatasi (Diptera: Psychodidae) lines susceptible and refractory to Leishmania major infection. American Journal of Tropical Medicine and Hygiene 42, 320-328. Zhang, W.W. and Matlashevski, G. (1997). Loss of virulence in Leishmania donovani deficient in an amastigote specific protein A2. Proceedings of the National Academy of Sciences of the USA 94, 8807-881 1. Zilberstein, D. (1991). Adaptation of Leishmania species to an acidic environment. In: Biochemical Protozoology ( G . Coombs and M . North, eds), pp. 349-358. London: Taylor and Francis. Zilberstein, D. and Shapira, M . (1994). The role of pH and temperature in the development of Leishmania parasites. Annual Review of Microbiology 48,449-470. Zuckerman, A. (1975). Current status of the immunology of blood and tissue protozoa. I . Leishmania. Experimental Parasitology 38, 370-400.


Immunity and Vaccine Development in the Bovine Theilerioses Nicola Boulter and Roger Hall* Department of Biology, PO Box 373, University of York, York, YO1 5 Y W , UK

Abstract ................................ ................................... 42 1. Introduction.. ........................ ................................... 43 1.1. Bovine Theilerioses .... ........................................... 43 1.2. Clinical and Pathological Features.. ...... ........................... 48 1.3. Control Measures .......................................... . . . . . . . 49 2 . Theileria annulata .......................................................... 51 51 2.1. Immune Responses .................................................... 2.2. Vaccination ............................................................ 62 3. Theileria parva ............................. ............................ 67 3.1. Overview .................................................. 3.2. Immune Responses.. ...................................... 3.3. Vaccination ............................................................ 73 4. Theileria sergenti. ........ .......................... 77 4.1. Classification . . . . . . . . .......................... 77 4.2. Clinical Features and Control .... .................... 4.3. Immune Responses.. . . . . . . . . . . . ................................... 78 4.4. Vaccination with Non-living Comp n t s . . .............................. 79 5. Comparative Aspects ....................................................... 80 6. The Future ............................... ............................ 82 Acknowledgements ............................................ . . . . . . . . . . 82 References. ........................... ..................................... 83

*Corresponding author. ADVANCES IN PARASITOLOGY VOL 44 ISBN 0- 12-031744-3

Cupl.rr~hr11.. 2000 Academic Press

A / / right., oJ' rrprodut rioi: in any liirnl rrs~~ri'ei!

42

N. BOULTER AND R. HALL

ABSTRACT There are three economically important bovine Theileria species: Theileriu annulata, which causes tropical theileriosis and occurs across north Africa and most of central Asia; Theileria purva, which causes East Coast fever and is found in East and Central Africa; and Theileria sergenti, which is predominantly a problem in Japan and Korea. Theileriu annulata preferentially infects macrophages in vivo. It is controlled largely by means of live, attenuated vaccines, which are produced by prolonged tissue culture of the schizont-infected cells. The immunity induced in animals, which have either recovered from an infection or have been vaccinated (with an attenuated vaccine), is broad, solid and cell mediated. It is considered that the main effector cells are cytostatic macrophages that produce nitric oxide. Subsidiary roles for bovine leucocyte antigen (BOLA)-restricted, transiently appearing, cytotoxic T cells, and possibly also natural killer (NK) cells, have been identified. Cytokines such as tumour necrosis factor a (TNF-a) may have important roles, particularly in the induction of pathology. Matrix metalloproteinases have been implicated in the metastatic behaviour of schizont-infected cells. The nature of the protective schizont target antigens remains unknown. Attempts to develop a subunit vaccine have focused upon a sporozoite antigen (SPAG-1) and a merozoite antigen (Tamsl). Both SPAG-1 and Tams I have given partial protection using different delivery systems and adjuvants, but further vaccine development will probably require identification of a range of other antigens, especially from the schizont stage. Theileria parva has a tropism for T cells. Vaccination is currently by the ‘infection and treatment’ method, which involves challenging with a controlled dose of sporozoite stabilate and the simultaneous administration of long-acting tetracyclines. The immunity thus induced is mediated by BOLA-restricted cytotoxic T cells, which recognize polymorphic schizont antigens. These antigens have not been characterized at the molecular level. However, the polymorphic nature of the target antigens underlies the fact that the immunity is very strain specific - a situation that distinguishes T. parva from T . annulata. Interestingly, it is not possible to produce an attenuated vaccine to T . parva, as T . parva requires up to two orders of magnitude more schizonts in order to achieve transfer to the new host. A suggested reason for this is that the macrophage targets of T. unnulata are phagocytes and thus the schizont has a natural, efficient route of entry whilst the preferred host of T . parva is the non-phagocytic T cell. Analysis of the cytotoxic T-cell response has revealed evidence of BOLA haplotype dominance plus competition between parasite epitopes. Subunit vaccination using a recombinant sporozoite antigen (p67) has proved very promising,

IMMUNITY IN THE BOVINE THEILERIOSES

43

with levels of protection of the order of 70% being achieved. A proportion of the protected calves exhibits complete sterile immunity. Interestingly, the basis for this immunity is not clear, since there is no correlation between the titre of antibodies that inhibit sporozoite penetration of lymphocytes and protection. Similarly, there is no significant T-cell response that distinguishes the protected and susceptible animals. These data are very encouraging, but other components, particularly those derived from the schizont, need to be identified and characterized. The mild Theileria species of Japan and Korea (termed T . sergenti in the literature) cause fever and severe chronic anaemia. The schizont stage of the life cycle is very rare and the host cell type is not known. The pathology is associated with chronic piroplasm infection. Immunity can be induced by immunizing with crude piroplasm extracts. Serological analysis of immune sera reveals that the immunodominant antigen is a polypeptide of 3033 kDa, which corresponds to the protective T . annulata polypeptide Tams1 . Passive immunity can be transferred with a monoclonal antibody to this 30-33 kDa molecule. Active immunization with a synthetic p32 antigen afforded protection against clinical symptoms and parasitaemia, confirming the importance of this antigen. A subunit vaccine against T . sergenti will therefore almost certainly include the 30-33 kDa antigen. As yet there is no subunit vaccine sufficiently protective against any of the Theileria species to allow widespread field usage. The focus of research, particularly for T . annulafa and T . parva, is the identification of the protective schizont antigens. The use of naked DNA delivery systems is also likely to be the method of choice, since the full spectrum of immunity, including T-cell responses, can be provoked. The vectors will most probably include selective immunopotentiators such as cytokine genes. It is likely that the vaccines that emerge will be multicomponent, being composed of several antigens from each life-cycle stage. Much of the research into vaccine development will, however, be empirical.

I. INTRODUCTION 1.1. Bovine Theilerioses

1.1.1. Background and Scale of the Problem

Theileria species are tick-borne protozoan parasites belonging to the phylum Apicomplexa. They are characterized by having an intracellular schizont stage that multiplies in leucocytes. This property distinguishes Theileria from true Babesia species, as the latter develop exclusively within

44


erythrocytes (Mehlhorn and Schein, 1984). There are many species of Theileria that affect domestic and wild animals (Mehlhorn and Schein, 1984; Irvin and Morrison, 1987; Dolan, 1989). For the purpose of this review, only three pathogenic species affecting cattle, T . annulata, T . parva and T . sergenti, will be considered in detail (Table 1). Theileria annulata (Dschunkowsky and Luhs, 1904) is transmitted by ixodid ticks of the genus Hyalomma, principally H . anatolicum anatolicum, and is the causative agent of the extremely debilitating and often fatal disease known as tropical theileriosis. Former synonyms include Mediterranean Coast fever, tropical gonderiosis and tropical piroplasmosis (Neitz, 1957). The true economic impact of tropical theileriosis is difficult to assess, but it threatens several hundreds of millions of cattle in a massive area encompassing North Africa, southern Europe, the Middle East and Central Asia, including the Indian subcontinent (Robinson, 1982). In India, where much effort is directed towards cross-breeding programmes aimed at increasing the milk yield of the national herd, potential losses as a result of tropical theileriosis are estimated to be US$ 800 million per annum or 10% of the gross national product (Brown, C.G.D., 1990; Devendra, 1995). In endemic areas, the indigenous cattle are usually infected as calves, when they show a milder clinical reaction. If they recover, they develop a persistent carrier state and become resistant to reinfection (Gautum, 1981; Brown, C.G.D., 1990). However, exotic breeds of cattle, particularly imported taurine dairy cattle, and cross-bred cattle are extremely susceptible, and the disease can cause mortality rates of 40-60% in these groups (Brown, C.G.D., 1990). Thus the need to develop efficient control measures against T. annulata is becoming ever more important as the practice of improving local dairy and beef herds by cross-breeding becomes more popular. Theileria parva causes the extremely virulent disease known variously as East Coast fever, Corridor disease or January disease. It is transmitted by the three-host tick Rhipicephalus appendiculatus in areas of East and Central Africa, where it is a major constraint on livestock production. The mortality for Bos taurus exotic breeds exceeds go%, whilst it is less often fatal but none-the-less extremely debilitating in local Boran cattle. Interestingly, it is only mildly pathogenic in the African buffalo, Syncerus caffer (see Irvin and Morrison, 1987). Total losses due to T . parva are estimated to be US$ 168 million per annum (Muhkebi, 1992). There is much confusion and debate over the nomenclature of the so-called benign Theileria species of Asia, Australia and Europe (Sugimoto et al., 1991, 1992; Kawazu et al., 1992a,b; Tanaka et al., 1993; Fujisaki et al., 1994; Katzer et al., 1994, 1998; Shiels et al., 1995; Kubota et al., 1996; Stewart et al., 1996). These are currently known as T . sergenti in Japan and Korea, T . buffeli in Australia and T. orientalis elsewhere (Fujisaki et al., 1994), although they are commonly collectively called the T . sergenti/orientalis/buffeli group. They are


45

all transmitted by ticks of the genus Haemaphysalis. Since the purpose of this review is principally immunological, not taxonomic, we intend to use the term T . sergenti, as this is how the Japanese and Korean species, upon which most of the relevant immunological work has been done, are described in the primary literature. It should be pointed out that T . sergenti is often pathogenic, but not in all cases, and rarely fatal (0.2-0.4% mortality), to ‘western’ breeds (e.g. Holstein) and less so to indigenous (Japanese Black) breeds, causing chronic anaemia and serious loss of productivity. The economic impact to Japan is estimated at US$15 million per year (Sugimoto, 1997). In passing, it is worth mentioning that the benign species of Theileria of Africa, namely T . mutans and T . velifera (also possibly found in the Caribbean), can be differentiated from the other benign species of Theileria as they are transmitted by ticks of the genus Amblyomma (see Young, 1990). Note also T . taurotragi, usually apathogenic but occasionally fatal, which infects cattle, sheep, goats, eland and other bovids in Africa and is transmitted by ticks of the genus Rhipicephalus (see Dolan, 1989). 1.1.2. Life Cycle The life cycle is essentially the same for all Theileria species and has been extensively documented elsewhere (Barnett, 1977; Mehlhorn and Schein, 1984; Higuchi, 1986, 1987; Morrison et al., 1986; Irvin and Morrison, 1987; Kawamoto et al., 1990; Tait and Hall, 1990; Stewart et al., 1996). Development of the parasite takes place within the vertebrate and invertebrate hosts, with asexual reproduction by schizogony and merogony * in the bovine host followed by sexual reproduction and sporogony in the tick vector. Theileria annulata sporozoites preferentially invade major histocompatibility complex (MHC) class I1 positive cells (monocytes and B cells) in vitro (Glass et al., 1989; Spooner et al., 1989; Campbell et a[., 1994) and the host cells in vivo are now established to be monocytes (Forsyth, 1997; Forsyth et al., 1997, 1999). In contrast, T . parva sporozoites can infect and transform B and T cells at similar frequencies in vitro, but the majority of parasitized cells in the tissues of infected cattle are cx/p T cells (Baldwin et al., 1988; Dolan, 1989; Morrison et al., 1996). The detailed phenotype of the host cell type(s) transformed by T . sergenti schizonts has not been documented. They are, however, very rare, a feature which clearly distinguishes T. sergenti from T . annulata or T . parva, and the host cell is very enlarged (Kawamoto et a[., 1990; Minami et al., 1990; Kawazu et al., 1991; Sato et al., 1993, 1994).

* ‘Schizogony’ refers to the macroschizont and ‘merogony’ to the microschizont.

P 0)

Table 1 Comparative aspects of Theileria annulata, Theileria parva and Theileria sergenti. Thderia parva

Theileria onnulata

Theileria sergen ti

Location

Southern Europe, North Africa, Middle East, Central Asia, India, China

East and Central Africa

Japan, Korea, possibly world-wide'

Tick vector genus

Hyalomma

Rkipicephalus

Haemaphysalis

Estimated cost per annum

US$800 million in India

US$168 million total

U S 1 3 million in Japan

Mortality to exotic

40-60Y0

> 90%

Rare

Host cell type invaded and transformed by sporozoitc in vivo

Macrophage/monocy te

T cell

Not known but resides in lymph nodes and liver

Stage of life cyclc causing most pathology

Schizont and piroplasm

(Bos taurus) cattle

Z m

0 C

i Schizont

Piroplasm

P I

Disease

Tropical theileriosis

East Coast fever

Japanese bovine theileriosis

F

I-

Major clinical and pathological features

Live vaccine

Listlessness, anorexia, cachexia, diarrhoea, leucopenia, dyspnoea, petechial haemorrhages in major organs, ulcerated lesions of the gut, widespread dissemination of schizont-infected cells to major organs, anaemia Yes. Attenuated macroschizonts. Made by long-term tissue culture. > 90% effective. Cross-protective

Listlessness, anorexia, cachexia, diarrhoea, leucopenia, dyspnoea, petechial haemorrhages in major organs, ulcerated lesions of the gut, widespread dissemination of schizont-infected cells to major organs. Most virulent of the bovine Theileria species

Chronic relapsing anaemia

No; but can use ‘infection and treatment’ method. Naturally avirulent strains may have potential. Protects against homologous challenge only

Yes. Blood vaccine has been used. Not currently recommended

r

I C z_

2Z -I

I rn

m _.

2z

rn -I

I

! r

rn

Main effectors in immune animals

Cytostatic macrophages. Transient BOLA-restricted CTL and non-restricted NK cells

BOLA-restricted CD8+ CTLs

Antibodies to the merozoite/piroplasm?

Progress in subunit/recombinant vaccine research

Partial protection with recombinant SPAG-1 (sporozoite surface) and Tams (merozoite surface) proteins in some trials

Complete protection in a proportion of animals and partial protection in a proportion using p67 (sporozoite surface) protein in several trials with a range of delivery systems. One trial showed cross-protection against heterologous stocks

Partial protection with recombinant p32/34 (merozoite surface) proteins in one trial. Passive protection using anti-p33/34 monoclonal antibody

’

-

Theileria sergenti is a taxonomically controversial term and hence it is difficult to comment accurately on its distribution. BOLA = bovine leucocyte antigen; CTL = cytotoxic T lymphocyte; NK = natural killer.

z $ rn v)

48


1.2. Clinical and Pathological Features

The clinical aspects of tropical theileriosis have been well characterized and documented (Neitz, 1957; Barnett, 1977; Srivastava and Sharma, 1981; Eisler, 1989; Forsyth, 1997; Forsyth et al., 1997, 1999). The course of the disease varies depending on the parasite strain, the host’s susceptibility and the quantity of sporozoites inoculated. It is well known that the severity of the disease is directly proportional to the initial inoculum of sporozoites (Uilenberg, 1981; Preston et al., 1992b). Time to detection of the initial symptoms varies from 4 to 14 days after the host is bitten by an infected tick. Swelling of the lymph node adjacent to the site of the tick bite is quickly followed by generalized lymphadenopathy. Schizonts can often be found in the lymph nodes at this time (Srivastava and Sharma, 1981). Hyperpyrexia is a hallmark of the disease and usually persists until death or recovery (Srivastava and Sharma, 1981). As the illness progresses the animal becomes anorexic, and there is a rapid loss of weight and condition. Other symptoms which accompany the disease are listlessness, mucous discharge from the eyes and nostrils, diarrhoea and dyspnoea. In all severe cases, petechial haemorrhages of the serous and mucous membranes occur and ulcers of the abomasum are also occasionally seen on post-mortem examination (Srivastava and Sharma, 1981). During the terminal stages there is usually oedema of the lungs, which causes severe respiratory distress, leucopenia, marked haemolytic anaemia, bilirubinuria, bilirubinaemia and jaundice (Neitz, 1957). The anaemia is thought to be a result of removal of erythrocytes by phagocytosis rather than parasite-induced lysis, although it has been suggested that autoimmune responses may contribute (Uilenberg, 1981). Tumour necrosis factor a (TNF-a) may also play a role (see Section 2.1.3, p. 56). Death usually occurs within 2-4 weeks from the onset of infection. Cattle that survive may undergo a prolonged period of recovery, which, in severe cases, may be incomplete, and animals may remain permanently debilitated and unproductive. In such cases a carrier piroplasm state ensues (Irvin and Morrison, 1987). The disease characteristics are similar for T. parva but some important differences must be noted. For example, in T.parva piroplasm parasitaemias are lower and anaemia is usually slight, with the main pathological effect arising from the lymphodestructive stage (Morzaria and Nene, 1990). In contrast, in other Theileria species, including T. sergenti, the severity of symptoms relates to the piroplasm infection rate and the resultant anaemia (Barnett, 1957; Jura and Losos, 1980; Morrison et al., 1986; Shimizu et al., 1990; Kawamoto et al., 1991; Yagi et al., 1991). T. sergenti causes chronic anaemia and one characteristic, resulting from a persistent carrier state, is relapsing parasitaemias, which can be induced by stress, and such episodes may be fatal.


49

Occasionally, T. parva, T. taurotragi and T. mutans infections are associated with a syndrome known as turning sickness (Irvin and Morrison, 1987). The condition is usually associated with the presence of large numbers of free schizonts and infected lymphoid cells in brain capillaries and large haemorrhagic necroses in cerebral tissues. Affected animals show nervousness, ataxia and circling and may die during convulsions in this form of the disease, which usually occurs several months, and even years, after the initial theilerial disease (Irvin and Morrison, 1987).

1.3. Control Measures

Current control measures against bovine theilerioses are fourfold: tick control; chemotherapy; vaccination with attenuated lines; and vaccination by ‘infection and treatment’ (Brown, C.G.D., 1990; Musisi, 1990; Singh, 1990; Tait and Hall, 1990; Pipano et al., 1991; de Castro and Newson, 1993; Stewart et al., 1996). Each method will be briefly discussed separately. 1.3.1. Tick Control The conventional method of tick control is using acaricide in dips or sprays (Chizyuka and Mulilo, 1990; Musisi, 1990). More recently, acaricideimpregnated ear tags, slow-release rumen boluses and ‘pour-ons’ have been used (de Castro and Newson, 1993). Although these latter methods are more ‘user friendly’, disadvantages still exist including cost, residual contamination of meat and milk, and acaricide resistance of the ticks (Young et al., 1988). Continuous use of acaricides can also result in the breakdown of anti-tick immunity, causing a loss of equilibrium or ‘endemic stability’, and leave the cattle more susceptible to infection (Tatchell, 1981; Young et a[., 1988; Pipano and Grewal, 1990). Problems of susceptibility also occur when highly productive dairy cattle are introduced into an endemic area. One solution is to maintain these herds as ‘tick free’, involving the confinement of cattle on pasture in isolation, or in yards or barns to which tick-free fodder is transported, under a regime of regular short-interval application of acaricides (Lawrence, 1990). T h s system is obviously very expensive and it is prone to breaking down with the subsequent infection of many, if not all, cattle. Resistance of cattle to ticks can be produced by controlled infestations and taurine cattle immune to Rhipicephalus appendiculatus have been documented (Cunningham, 1981). Vaccination against many species of ticks, using crude antigenic extracts, is partially successful (reviewed by Kay and Kemp, 1994). A significant recent development is the production of a commercial subunit vaccine against Boophilus microplus, the vector for Babesia bigemina and

50


B. bovis (see Cobon and Willadsen, 1990; Willadsen, 1990; Willadsen et al., 1995). A similar vaccine against the ticks responsible for the transmission of any of the bovine Theileria species would obviously be a useful control tool. 1.3.2. Chemotherapy Chemotherapy has not been widely used for the treatment of T. annulata, but it is an important component in the control of T. parva and T. sergenti infections (Purnell and Chang Rae, 1981; Stewart et al., 1990a,b; Tait and Hall, 1990; Hagiwara et al., 1993; Stewart et al., 1996). Halofuginone, a febrifugine, and the hydroxynapthoquinones parvaquone and buparvaquone, are all effective against Theileria infections (McHardy et al., 1985; Stewart et al., 1990a,b; Hagiwara et al., 1993). Buparvaquone has much higher anti-theilerial activity than parvaquone (Hashemi-Fesharki, 199la). It appears that the actions of these drugs are different; parvaquone is active against all stages of Theileria, but halofuginone and buparvaquone are active only against the schizont stage (Young et al., 1988). Halofuginone, despite being relatively cheap, is no longer a preferred drug because it has a narrow therapeutic range. Oxytetracyclines are also used to limit parasite development in the so-called ‘infection and treatment’ method as described in Section 1.3.4 below. 1.3.3. Vaccination with Attenuated Parasites The most widespread control measure against T. annulata is the inoculation of animals with an attenuated cell line vaccine. This involves inoculation of schizont-infected cells derived from a continuously growing tissue culture in vitro and is discussed in detail in Section 2.2.1 (p. 62). The production and use of schizont tissue culture vaccines has been described by many authors including Pipano (1981), Hall (1988), Brown, C.G.D. (1990) and Tait and Hall (1990). The cell culture vaccine protects most breeds of cattle against homologous challenge and often against heterologous challenge (Pipano, 1981). A single vaccination is usually sufficient but exotic breeds may require a second vaccination with a heterologous strain to provide full protection. A blood vaccine has been used against T. sergenti but it is no longer recommended. 1.3.4. Vaccination by ‘Infection and Treatment’ Another method of vaccination is the infection and treatment method (discussed in detail in Section 3.3.1, p. 73). This method was initially


51

designed for use against T. parva and, as the name suggests, cattle are deliberately infected with a defined dose of sporozoites and then treated with a chemotherapeutic agent (Morzaria and Nene, 1990). Long-acting oxytetracyclines are the drugs of choice for this method when applied to T . parva, but buparvaquone is preferred in the case of T. annulata (see Hashemi-Fesharki, I99 la). The timing of treatment is important. Giving chemotherapy immediately after infection might clear the parasites before they have had the chance to establish themselves in the host lymphocytes, resulting in a lack of immunity to challenge. On the other hand, chemoprophylaxis must be given early enough to prevent the development of clinical symptoms (Morzaria and Nene, 1990). This type of vaccination provides solid immunity to challenge with homologous parasites and, depending on source of infection, frequently against heterologous challenge. 1.3.5. The Need for New Vaccines

There are several actual and theoretical problems associated with the use of live parasites. The infection and treatment method uses virulent stocks of parasites for the immunization regime. T h s method also results in the presence of piroplasms in the immunized animals, which can aid the spread of the disease by ticks to unprotected cattle (Tait and Hall, 1990). In addition, the drugs used are very expensive. Reversion to virulence of attenuated parasites used in the cell culture vaccine is always a worrying possibility, but there has been no indication of this happening so far. Drawbacks related to the use of either vaccine involve the need for a ‘cold chain’ from the point of production to the location of use, as well as the potential for transmitting other pathogens with the immunizing parasites (Tait and Hall, 1990). All of these problems could be avoided if non-living, effective, stable, cheap, single application subunit vaccines for these pathogens could be developed. To achieve this objective requires a combined molecular, genetic and immunological approach. A balance between fundamental and empirical research is also required. Eventually the ‘new’ safer vaccines will form part of integrated disease control programmes (Young et al., 1988).

2. THElLERlA ANNULATA

2.1. Immune Responses

The bovine immune system is subjected to different antigenic determinants at each stage of the parasite’s life cycle, and this results in a heterogeneous

52


nocyte/macrophage

Neutralizing

, +

e.g. anti-SPAG-1 antibodies

f

% @ Invasion

1 Cytostatic macrophage

I

development 24 hours

TNF-a and IL-ip, etc

.

._.._I._

Inhibition by cytostasis

4

antibodiese.g. anti-p32/33

Figure 1 Summary of the proposed immune response against Theileria annulata. The figure is drawn to emphasize the proposed distinctive roles for the innate immune response (on the right) and the adaptive immune response (on the left). The thickness of the arrows is intended to reflect the relative importance of the relevant immune mechanisms. The major effector depicted is the cytostatic macrophage and the prime effector mechanism is nitric oxide-mediated inhibition of the schizont-infected macrophages. Note that TNF-a inhibits development from the trophozoite to the


53

response comprising humoral and cellular components. In all cases, animals that recover from infection are solidly immune to homologous challenge and often to heterologous challenge (for a review, see Hall, 1988). Immunity is acquired irrespective of the mode of primary infection, whether this be by the natural route via injection of sporozoites by a feeding tick, or by artificial immunization with sporozoites followed by chemotherapy, or by vaccination with attenuated schizont-infected cell lines (Pipano, 198 1). Immunity has been shown to last for approximately 3 years or longer if the animal is challenged (Pipano, 1995). The immune response to each stage of the life-cycle will be discussed separately and the overall situation is summarized in Figure 1 and by Preston et al. (1999). 2.1.1. The Sporozoite From the outset it should be stated that the very existence of an effective attenuated schizont vaccine (see Section 2.2.1, p. 62) means that immunity to the sporozoite stage is not essential to achieve protection. On the other hand, even a partially protective response against sporozoites might well prove beneficial by reducing the severity of the ensuing disease. This line of thinking is based on the established correlation between sporozoite dose and virulence of the disease (Preston et al., 1992b) plus the fact that live sporozoite immunization provides better protection against heterologous challenge than do attenuated cell lines (Sergent et al., 1945; Preston and Brown, 1988). Since the sporozoite is extracellular, intuition suggests that effective anti-sporozoite immunity might be humoral, and specifically might be mediated by neutralizing antibodies. Evidence for such antibodies comes from the work of Gray and Brown (1981), who clearly demonstrated that sera taken from animals, after infection with a stabilate of T. annulata sporozoites (either Hissar or Ankara strains), contained activity that neutralized the infectivity of sporozoites for bovine peripheral blood mononuclear cells (PBMs) in vitro. These sera were also capable of inhibiting invasion by sporozoites from a heterologous parasite stock, which suggests that parasites from geographically distinct regions share some common antigenic determinant(s). It should, however, be pointed out

schizont stage but not the multiplication of schizonts. Natural killer cells also play a role in lysing the schizont-infected macrophage. Nitric oxide is also able to inhibit the ability of sporozoites to invade the host cell in vitro. In the adaptive immune response, a central role for BOLA-restricted cytotoxic T-cells targeted against the schizontinfected cells is depicted. Neutralizing antibodies against the sporozoite and merozoite stages are also thought to be important. APC = antigen presenting cell; CTL = cytotoxic T lymphocyte; IL = interleukin; M@ = macrophage/monocyte; NK = natural killer cell; NO = nitric oxide; rbc = red blood cell.

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that these experiments used whole serum and thus do not formally prove a role for antibodies. Proof of antibody-mediated sporozoite neutralization is provided by the demonstration that several different monoclonal antibodies (mAbs) to sporozoite antigens can also abrogate infectivity. One such mAb, 1A7, blocks sporozoite invasion effectively and also reacts positively in immunofluorescent (IFA) tests against formalin-fixed sporozoites, but not against the schizonts or piroplasms (Williamson, 1988; Williamson et al., 1989). mAb 1A7 also recognizes sporozoites from three geographically distinct stocks (from Morocco, India and Turkey), which are known to differ by several other criteria, including reactivity to a panel of anti-schizont mAbs and genotypically (Shiels et al., 1986; Katzer et al., 1999). Recent work has focused on characterizing and assessing the immunological significance of the sporozoite antigens defined by the neutralizing mAbs (Knight, 1993; Boulter et al., 1995; Knight et al., 1996, 1998). The gene for the sporozoite surface antigen (SPAG-1) of T. annulata, recognized by mAb lA7 and derived from a T. annulata Hissar complementary deoxyribonucleic acid (cDNA) library, has been well characterized. SPAG- 1 has been shown to be located on the surface of the sporozoite by immunogold electron microscopy (Knight, 1993). There is a single open reading frame extending for 2721 nucleotides encoding a predicted 91.9 kDa polypeptide of 907 amino acids (Hall et al., 1992). A number of structural and immunological features are summarized in Figure 2. Perhaps of most interest are two blocks of repetitive motifs of PGVGV and VGVAPG (single-letter amino-acid notation). The former of these is identical to repeat structures found in bovine elastin. The latter is also found in elastin and has been demonstrated to be chemotactic and a ligand for the elastin receptor (Davidson, 1987). The presence of the VGVAPG motifs led to the suggestion that they might be involved in host cell recognition (Hall et al., 1992). However, this idea has since been weakened by the discovery that some variants of SPAG-1 lack the VGVAPG motif and also that sporozoites invade cells lacking the elastin receptor (Campbell et al., 1994; Hall, 1994; Katzer et al., 1994). There are several immunodominant sites on the SPAG-1 molecule, as determined by reactivity to various subfragments of SPAG-1 in Western blots with a panel of bovine immune sera, and these are summarized in Figure 2 (Boulter, 1996; Knight et al., 1996). Furthermore, SPAG-1 has been shown to carry neutralizing determinants in the C-terminus defined by immune calf sera, which are distinct from the 1A7 epitope (Williamson, 1988; Williamson et al., 1989; Hall et al., 1992; Boulter et al., 1994, 1995; Boulter, 1996). Partial protection has been achieved with recombinant SPAG-1 (Boulter et al., 1995, 1998, 1999) and this will be described in detail in Section 2.2.2 (p. 64). In addition to antibody responses, SPAG-1 has also been shown to elicit T-cell responses, although these are very variable


55

Figure 2 Comparison of the sporozoite surface proteins from Theileria annulata (SPAG-1) and T . parva (p67) shown diagramatically to emphasize their structural and immunological similarity. The numbers above the boxes refer to amino-acid residues in SPAG-1. The anti-SPAG-1 and anti-p67 epitopes were defined by reacting bovine antisera (to recombinant SPAG-1 and p67) with short regions of SPAG-1 (Knight et al., 1996).

(Boulter ef al., 1995, 1998, 1999). Recent experiments have shown that extremely strong CD4+ memory T-cell responses can be generated to the N terminus of the SPAG-1 molecule, in the presence of interleukin 2 (IL-2) from the PBMs of immune animals (J.D. Campbell, personal communication). T-cell clones, specific to the N terminus, have now been generated and are the first T cells to be isolated in vitro specific for a T. annulata-derived peptide. An interesting observation is the cross-reactivity of SPAG-1 and the T. parva homologue, p67 (Figure 2; Knight et al., 1996). There is a 56% identity between the C-terminal regions of SPAG-1 and p67 (Figure 2; Katzer et al., 1994). mAb 1A7, which reacts to a C-terminal epitope of SPAG-1, cross-reacts with p67 and is also capable of neutralizing T. parva sporozoite infectivity with 100% efficiency. In addition, seven continuous amino acids from the predicted sequence recognized by lA7 are present in the p67 sequence and would be sufficient to form a common epitope (Knight et al., 1996). The potential importance of the C-terminal regions of these

56


molecules is supported by the evidence that antisera raised against recombinant p67 block T . annulata sporozoite invasion in vitro and vice versa. Furthermore, vaccination trials in which animals have been immunized with SPAG-1 and challenged with T . parva sporozoites, or immunized with p67 and challenged with a T. annulata sporozoite stabilate, provide some evidence of cross-protection to the heterologous challenge (Boulter et al., 1998, 1999, unpublished data; see Section 2.2.2, p. 64). Hence there is a possibility of designing a subunit vaccine containing either SPAG-1 or p67, which will be effective against both T . annulata and T.parva sporozoites. Two different antigens (called SPMl and SPM2), both recognized by sporozoite-neutralizing mAb 4B11, have been characterized (Knight, 1993; Knight et a[., 1998). Unlike SPAG-1, these two antigens are also expressed on the schizont and piroplasm stages and, other than being recognized by immune calf serum, their protective relevance remains obscure. Apart from antibodies, nitric oxide (NO), produced by activated macrophages, has been shown to inhibit sporozoite invasion into host cells in vitro (Visser et al., 1995). Whether this is effective in vivo remains to be elucidated. 2.1.2. The Trophozoite Theileria trophozoites are susceptible to factors in immune serum (Preston and Brown, 1985). Thus when cultures, containing parasites just established within the host cells, were incubated with T . annulata immune sera, the development of trophozoite-infected cells into schizont-infected cells was suppressed (Preston and Brown, 1985). Initially, it was postulated that this activity was antibody-dependent and was mediated via sporozoite antigens left on the surface of the invaded lymphocyte after entry, but further work indicated that it is more likely that other serum factors, probably cytokines, are responsible (Preston et al., 1992a). To this end, a comprehensive study of the effect of various recombinant cytokines on the development in vitro of T . annulata and T. parva trophozoite-infected cells showed that TNF-a, interferon y (IFN-y), IFN-a, IL-1 and IL-6 were inhibitory (Figure I; Preston et al., 1992a). However, the mode of inhibition by these cytokines remains obscure, although they may be inducing NO production (Visser et al., 1995).

2.1.3. The Schizont (a) Protective immunity. Cell-mediated responses to the schizontinfected cell are generally thought to be responsible for protective immunity as well as for much of the underlying pathology (Rehbein et al., 1981a,b;


57

Preston et al., 1983; Ahmed et al., 1989; reviewed by Tait and Hall, 1990; Kachani and Spooner, 1992; Chaudhri and Subramanian, 1992). Currently, both the innate and adaptive arms of the immune system are believed to play important effector roles. In particular, roles for cytotoxic T cells, natural killer (NK) cells, helper T-cells and macrophages have been identified and are elaborated below (Figure 1). Preston et al. (1983) demonstrated that, when immune cattle are challenged, two peaks of cytotoxic cell activity were observed in the blood and lymph nodes. The cells in the first of these peaks, appearing one week after challenge, were bovine leukocyte antigen (BoLA) (MHC class I) restricted, whereas those in the second peak, observed three weeks after challenge, were BoLA restricted in some, but not all, animals. The MHCrestricted components are cytotoxic T-cells (CTLs), whilst the non-restricted class are designated as NK cells. In the same study it was shown that calves undergoing a primary infection, but destined to recover, also developed two peaks of cytotoxic cells. In contrast, calves that did not recover rarely showed such responses. In a more recent study, Chaudhri and Subramanian (1992) produced similar but not identical results. They demonstrated that three calves that survived a primary infection induced by sporozoites, or a virulent schizont-induced infection, developed a single wave of cytotoxic cells that killed schizont-infected autologous cells. These reached a peak three weeks after infection, which correlated with a peak of T cells defined by the ability to form E rosettes with sheep erythrocytes. Calves that failed to recover from the primary infection had no, or only very weak, cytotoxic responses. A second peak of cytotoxic cells was induced after sporozoite challenge. In addition, T cells from these calves produced macrophage migration inhibition factor (MIF), in response to both schizont and piroplasm antigens. Innes et al. (1989b) showed that animals infected with an allogeneic T. annulata cell line exhibited very mild clinical reactions compared to the very severe response in animals infected with an autologous cell line. In the allogeneic group, there were two distinct waves of cytotoxic activity, the first being directed against the foreign BoLA antigens and the second aimed at the parasite. However, animals infected with the autologous cell line developed cytotoxicity only against the parasite, as expected. In both cases the anti-parasite response involved both BOLA-restricted and non-restricted elements. All cattle became immune to heterologous sporozoite challenge, during which they developed a cytotoxic response, which was MHC restricted and cross-reactive. The non-restricted cytotoxic response seen in T. annulata infections has been postulated to be due to NK cells (Preston et al., 1983). NK cells have been implicated as effectors in other intracellular parasitic infections such as those due to Trypanosoma cruzi (see Cardillo et al., 1996) and Leishmania

58


(see Tizard, 1992). They are characterized as large, granular, non-adherent, non-phagocytic, non-T and non-B cells that originate in the bone marrow and will kill xenogeneic tumour cell lines (Tizard, 1992). In model systems, NK cells are powerful producers of numerous cytokines including IFN-y, granulocyte-macrophage colony-stimulating factors (GM-CSFs), TNF-a, IL-3 and IL-8 (reviewed by Scott and Trinchieri, 1995). The activities of NK cells (cytotoxicity, proliferation and production of cytokines) are regulated by various cytokines. It is believed that NK cells usually become effective soon after an infection because many pathogens stimulate the production of cytokines critical for NK cell activation. The most powerful NK cell enhancers are IL-12, IFN-a and IL-2 (Scott and Trinchieri, 1995). In this regard it is interesting that schizont-infected cells have been shown directly to produce IFN-a (Entrican et al., 1991; Forsyth et al., 1999). However, interestingly, in the work of Preston et al. (1983) cited above, contrary to dogma, the adaptive BoLA-restricted CTL response preceded the innate NK response. Other innate effectors of the immune system are involved in protection against T. annufata, including cytostatic macrophages, which are believed to have an absolutely pivotal role (Figure 1). Preston (1981), Preston and Brown (1988) and Preston et al. (1993) clearly showed that macrophages, isolated from the peripheral blood of calves immunized with sporozoites or schizont-infected cell lines, exhibited strong cytostatic effects on schizontinfected cells. These cytostatic cells were active against allogeneic and autologous cell lines. Macrophage activity declined after a few weeks but rose again after challenge, with the same kinetics as before. The macrophages were shown spontaneously to produce TNF-a (Preston et af., 1993) and NO (Visser et af., 1995), and the levels produced were enhanced upon exposure of the cells to IFN-y, which suggests a role for antigen-specific helper CD4+ T cells. The cytostasis was shown to be mediated via a soluble factor, which was not TNF-a, IL-1 or IFN-a, and was probably NO, as discussed below (Preston et al., 1992a). In fact recombinant TNF-a (and IL-2) consistently enhanced the proliferation of schizont-infected cells (Preston et al., 1992a). This suggests that these cytokines may play a role in the pathogenesis of the disease (see below). The synthesis in vivo of IFN-y and TNF-a by host cells in response to T. annulata infection has been demonstrated (Preston et al., 1993). NO is a reactive metabolite of leucocytes, and in particular macrophages, and has been implicated in immunity to many protozoa such as Leishmania species (Tizard, 1992). The production of NO by these cells is promoted by IFN-y and TNF-a, and thus Visser et al. (1995) undertook a study to see if NO could be a mediator of macrophage anti-Theileria activity. PBMs from calves undergoing an infection spontaneously produced NO in vitro. However, PBMs from immune calves did not produce NO unless exposed


59

to IFN-y, indicating that NO production and release depend upon macrophages being both primed and triggered (Visser et a/., 1995). PBMs from an immune calf were stimulated to produce NO by co-cultivation with schizont-infected cells. These results, together with the fact that schizontinfected cells can stimulate lymphocytes to secrete IFN-y and TNF-a in vivo, suggested that these infected cells may initiate the production of macrophage-derived NO (Visser et a/., 1995). NO has been implicated as an effective mediator of protective immunity, in vivo, against trophozoite- and schizont-infected cells (Visser et al., 1995). Furthermore, NO has been shown by Richardson et al. (1998) to inhibit the proliferation of schizontinfected cells and to cause intracellular schizonts to disappear and host cells to become apoptotic. By extrapolation, these authors suggested that merozoites may also be susceptible to the effects of NO. In contrast to this protective role, it is postulated that NO, in excessive amounts, could well play a prominent role in the pathogenesis of Theileria; there is evidence that it plays a role in the pathogenesis of malaria (Mendis and Carter, 1995). (b) Immunopathology. It is well established that inappropriate immune responses can lead to pathology and disease. Recent evidence has indicated that primary infection with T. annulata induces aberrant T-cell activation, which in turn results in a failure to mount an effective immune response (Campbell et al., 1995, 1997; Campbell and Spooner, 1999). The consequence of this is an uncontrolled acute lethal infection. A manifestation of this inappropriate T-cell activation is the fact that T. annulata schizont-infected cells are able to induce non-specific proliferation of autologous resting T cells in vitro [dubbed the 'autologous mixed lymphocyte reaction' (MLR)] in the absence of added antigen (Preston, 1981; Campbell ef a/., 1995). In fact, this type of autologous MLR in vitro was first described for T. parva by Pearson et al. in 1979, but it has not been implicated in the pathogenesis of East Coast fever to date. Other indicators of naive T-cell activation by T. annulata-infected cells are the induction of IL-2 receptor (IL-2R) and MHC class I1 molecules on the surface of CD4+ and CD8' T cells from naive animals (Campbell et al., 1995).This activation appears to be mediated via a combination of direct cell contact and cytokine effects. Thus in vitro-derived schizont-infected cells express messenger ribonucleic acid (mRNA) for IL-la, IL-ID, IL-6, IL-10, TNF-a (Brown, D.J. et af., 1995) and IL-12 (J.D. Campbell, personal communication) and the levels of IL- 1a and IL-6 correlate with the level of induced proliferation. Resting CD4+ or CD8' T cells are stimulated by schizont-infected cells irrespective of their overall phenotype or memory status, although contactdriven activation is directed only at CD4+ cells (Campbell et al., 1997). Both cup and yS T cells are activated by schizont-infected cells. The autologous MLR phenomenon has been likened to a superantigen effect but there are differences. In particular, the proportion of T cells activated is much greater

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in the autologous MLR than would be expected for a superantigen, so this may be an entirely different phenomenon. According to Campbell, the counterpart in vivo of the autologous MLR results in the following sequelae (Campbell and Spooner, 1999). First, within 48 hours, large numbers of infected macrophages develop within the medulla of the lymph node draining the site of infection. Macrophages are professional antigen-presenting cells and, when infected with T. annulata, their capacity to present third-party antigens, via MHC class 11, to antigenspecific CD4+ T cells is augmented (Glass and Spooner, 1990). As a result, within 4 days, large numbers of CD4+ T cells are inappropriately activated in the medulla instead of the paracortex of the lymph node, where normal T-cell interactions should occur; i.e., during infection, T cells are not being primed in normal anatomical sites. Thus, instead of antigen-driven selection of a small number of T cells that would subsequently undergo clonal expansion, there results a rapid polyclonal expansion of IL-2Rf T cells that also quickly leave the node. The overall effect is ablation of the ability to generate an effective immune response. Additionally, it has been shown in some cases that infection with T. annulata results in a modification of the BOLA class I antigens on the surface of the transformed host cells (Oliver and Williams, 1996). These authors postulated that this may be a mechanism that the parasite uses to evade the host immune response. TNF-a may have a major role in the pathogenic effects (pyrexia, cachexia, anorexia, depression and anaemia) seen in T. annulata infection (Preston et al., 1993; Brown et al., 1995; Adamson and Hall, 1997; Forsyth et al., 1999). In fact, many of these symptoms are produced in cattle administered recombinant TNF-a (Bielefeldt Ohman et al., 1989). Macrophages harvested from cattle undergoing a primary or challenge infection of T. annulata spontaneously produce TNF-a and the levels produced are enhanced by exposure to IFN-7 (Preston et al., 1993). Abnormally high levels of IFN-y are seen in lethal T. annulata infections and thus may result in excessive amounts of TNF-a being produced (Campbell et al., 1997). Also high levels of matrix metalloproteinases produced by schizont-infected cells may enhance the conversion of pro-TNF-a to the mature species (Adamson and Hall, 1997). Furthermore, 76 T cells, which are stimulated to proliferate by autologous T. annulata-infected cells in the presence of IL-2, express an mRNA transcript for TNF-(u (Collins et al., 1996). TNF-a acts directly on the temperature-regulating centre in the hypothalamus to cause a fever (Tizard, 1992). This in turn may help the parasitic transition from schizonts to merozoites (Glascodine et al., 1990). Weight loss and cachexia in animals suffering from a chronic parasitic infection can be attributed, at least partly, to this cytokine. It inhibits the synthesis of lipoprotein lipase, acetyl coenzyme A, carboxylase and fatty acid synthetase. As a result it prevents the uptake of lipids by preadipocytes and causes mature adipocytes to lose


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stored triglycerides. TNF-a also acts on muscle cells and hepatocytes to stimulate their catabolism. In addition, TNF-a suppresses haematopoietic progenitors and thus decreases red blood cell production, and also reduces their life span, resulting in anaemia (Tizard, 1992). TNF-a has also been implicated in both the pathology (cerebral malaria, anaemia and fever) and protection (control of parasite multiplication) in Plasmodium infections (reviewed by Taverne, 1994; Jakobsen et al., 1995; Mendis and Carter, 1995), and in the cachexia associated with Trypanosoma cruzi infection in mice (Truyens et al., 1995). The pathology of tropical theileriosis is partly associated with the metastatic behaviour of the schizont-infected cell and the lesions induced in the invaded tissues (Forsyth et al., 1999). It is quite likely that the expression of matrix metalloproteinases induced by this organism are necessary, but probably not sufficient, to explain the mechanisms of tissue invasion and tissue damage (Baylis et al., 1992, 1995; Adamson and Hall, 1996, 1997; Somerville et al., 1998a). 2.1.4. The MerozoitelPiroplasm The merozoite, like the sporozoite, is an extracellular stage and is therefore also a potential target for a protective humoral immune response. Work on the merozoite stage has been hampered because of difficulties in obtaining sufficient amounts of merozoites for molecular analysis and the lack of an assay in vitro for erythrocyte invasion. The former problem has now been overcome by utilizing the fact that schizont-infected cell lines can be induced to differentiate into merozoites by culturing at 41 "C (Glascodine et al., 1990). This differentiation is associated with a change in mAb reactivity profile, with epitopes being detected on the merozoite and piroplasm which were absent from the schizont. One of these stage-specific antigens, recognized by mAb 5E1, was shown to have a molecular mass of 30 kDa (Glascodine e f al., 1990). This antigen was further characterized by Dickson and Shiels (1993). It was strongly recognized by serum from an immune cow and was shown to exist in two forms with molecular masses of 30 kDa and 32 kDa. The two molecules were shown to be related, and are in fact the products of alternative alleles, but only the 30 kDa form was recognized by mAb 5E1. Molecules with similar characteristics have been identified in other species of Theileriu with molecular masses ranging from 30 to 34 kDa (reviewed by Dickson and Shiels, 1993). The genes encoding for the 30132 kDa proteins from T . annulata, Tamsl-1 and Tamsl-2, respectively, have been isolated and characterized (Shiels et al., 1994, 1995). Sequence analysis of many alleles of Tams have shown that they encode very polymorphic molecules, particularly within a region that

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contains a number of putative N-linked glycosylation sites (Katzer et al., 1999). It is postulated that this level of antigenic diversity may indicate selection of variable glycosylation sites or amino-acid epitopes in order to evade the bovine immune system (Shiels et al., 1995). A number of other polypeptides has been immunoprecipitated from surface-iodinated piroplasms by immune cow serum (Shiels et al., 1989). These polypeptides probably originate from the merozoite and give some indication that an immune response might be mounted to this stage. In addition, Ahmed et al. (1988) provided evidence that immune serum contained antibodies that specifically opsonized free merozoites. Antibodyindependent complement lysis was also shown to occur. Thus, to summarize, infection with T. annulata stimulates both the innate and adaptive arms of the immune response. Activated macrophages producing TNF-a and NO, NK cells, cytotoxic T cells and antigensensitized CD4+ cells are all at work as the disease progresses. It is likely that the cytokines produced by the parasitized cells themselves modulate the immune response. However, there is a delicate balance between the induction of a protective and a pathological response. These considerations have important implications in subunit vaccine design. It is expected that the most effective immunity will be induced by vaccines that are able to stimulate CD4+ T-cell-mediated macrophage activation (Richardson et al., 1998). The inclusion of antigens that promote specific macrophage activation should result in the adaptive immune response being driven towards a protective Thl response and the generation of cytotoxic T cells, cytokines (predominantly TNF-a, and controlled levels of IFN-y) and NO, which have parasiticidal properties. 2.2. Vaccination

2.2.I . Attenuated Cell Line Vaccine The most widespread control measure taken against T. annulata is the vaccination of animals with an attenuated cell line vaccine. This involves inoculation of cattle with schizont-infected cells derived from a continuously growing tissue culture in vitro. The production and use of schizont tissue culture vaccines have been described by many authors including Pipano (1981), Hall (1988), Brown, C.G.D. (1990) and Tait and Hall (1990). Long-term culture attenuates schizont-infected cells so that their pathogenicity is reduced but their infectivity is retained (Darghouth et al., 1996; Sutherland et al., 1996; Somerville et al., 1998b). In addition, the ability to produce the merozoite stage is reduced both in vivo and in vitro once the cell line is attenuated. The number of passages required to achieve


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attenuation is dependent on the isolate: some require as few as 20 passages whilst others require up to 300 passages. Virulence is tested periodically by inoculating the culture into susceptible cattle (Hall, 1988). Once attenuation has been achieved, aliquots of the infected cells can be cryopreserved in liquid nitrogen. These can then be resuscitated and subcultured or used directly for immunization (Pipano, 1981), although the shelf-life of the infected cells once thawed is limited. This vaccine is effective because the schizont establishes itself in the recipient cells, an essential requirement if it is to engender protective immunity. The mechanism by which the schizont transfers from the donor to recipient cells is unknown, but Forsyth et al. (1997) suggested a process involving opsonization with complement. Schizont-infected cells express CDI Ib, the membrane receptor for C3bi, which is phenotypically a reflection of their macrophage origin. C3bi is a component of the complement cascade and an opsonin. It is suggested that free schizonts are opsonized by complement and then transferred to macrophages bearing the C3bi receptor. Attachment of the parasite-complement complex would thus enable phagocytosis of the parasite by the cell. Innes et al. (1989a) have shown that as few as 100 allogeneic cells can be used to immunize against T. annulata, whereas very high doses of allogeneic cells (> lo8) are required to immunize against T. parva. The normal vaccine dose is about lo6 cells. Since both parasite species are introduced in allogeneic cells, histoincompatibility between cell line and recipient cannot be the main influence on the successful establishment of the parasite within the new host. The different outcomes are probably a result of their preferences for different cell types and, specifically, the fact that T. annulata shows a distinct macrophage/monocyte tropism (Glass et al., 1989; Spooner et al., 1989). The mechanism of attenuation is not known and studies are currently under way to attempt to define the markers of attenuation. A number of genes has been identified that are either upregulated or downregulated on attenuation (Somerville, 1997; Somerville et al., 1998b). In addition, marked changes in the host matrix metalloproteinase profile of T. annulata-infected cells have been observed in some cell lines (Baylis et al., 1992, 1995; Somerville, 1997; Somerville et al., 1998b). Another interesting observation is the selective expression of the schizont antigen recognized by mAb EU106 (Sutherland et al., 1996). The antigen is stage-specific and is also expressed on the surface of infected host cells (Preston et al., 1998), but only in virulent cell lines; the expression diminishes upon attenuation. Attenuation, at least in some instances, is accompanied by clonal selection of a minor subpopulation or single genotype and/or altered parasite gene expression (Baylis et al., 1995; Darghouth et al., 1996; Sutherland et al., 1996; Somerville, 1997; Preston et al., 1998; Somerville et al., 1998b; Hall et al., 1999). The cell culture vaccine protects most breeds of cattle against

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homologous challenge and usually against heterologous challenge also (Pipano, 1981). A single vaccination is usually sufficient but exotic breeds may require a second vaccination with a heterologous strain to provide full protection (Nichani et al., 1997). The schizont vaccine has the advantage that it does not result in the production of erythrocytic stages in the immunized animal and therefore does not allow further transmission (Tait and Hall, 1990). However, it does not induce sterile immunity and cannot prevent the formation of piroplasms after natural tick challenge, and cannot therefore be used as a means of eradicating the disease (Pipano et a[., 1991). Nevertheless, this type of vaccine has been used successfully against T. annulata infection in many countries. These include: Israel, where it has been used for over 30 years (Pipano, 1995); Iran, in which a long-term vaccination campaign has been undertaken (Hashemi-Fesharki, 1988, 1991b); India, where the vaccine is produced commercially (Singh et al., 1993); the former Soviet Union (Zablotsky, 1991); and Turkey (Sayin et al., 1997). It is in various stages of development in other countries such as Iraq (Khdier and Latif, 1991), Tunisia (Darghouth et al., 1996, 1997), Morocco (Ouhelli et al., 1997), China (Gu et al., 1997; Shirong, 1997) and, most recently, Spain (Viseras et al., 1998). 2.2.2. Subunit Vaccine Development (a) General considerations. Although the cell line vaccines are highly efficacious, a number of drawbacks compromises their use (see Section 1.3.5). Testing and the long culture period needed to produce a vaccine, which make it expensive, are limitations. It is also unknown whether the parasite can revert to virulence in vivo. Furthermore, there are concerns about the likelihood of transmitting other pathogens with the immunizing parasite and there is also a requirement for a ‘cold chain’ from the point of production to the location of use (Tait and Hall, 1990). As a result of these limitations, research is currently aimed at producing a recombinant subunit vaccine that will obviate the problems. It is anticipated that such a vaccine will comprise components of the three major life-cycle stages, i.e. the sporozoite, schizont and merozoite/piroplasm. To date, vaccine trials in cattle have been performed with two recombinant antigens: SPAG-1, a sporozoite surface antigen (Hall et al., 1992), and Tams1 from the merozoite (Shiels et al., 1994, 1995). In most cases the recombinant antigens induced partial protection to sporozoite or blood challenge, respectively, but the trials showed that efficacy depended on the delivery system. (b) SPAG-1 trials. Five SPAG-1 trials have been performed to date, with various outcomes (Williamson, 1988; Knight, 1993; Boulter et al., 1995,


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1998, 1999). The first two, utilizing the C-terminal domain of SPAG-1 (SRl region, Figure 2) as a P-galactosidase fusion and the full-length SPAG-1 molecule expressed as a glutathione-S-transferase fusion, respectively, yielded disappointing results in that there was little evidence of protection against sporozoite challenge (Williamson, 1988; Knight, 1993; Boulter et al., 1999). However, sporozoite neutralizing antibodies were obtained and this observation gave the encouragement needed to investigate alternative ways of delivering the antigen in order to enhance the protective immune response. The first trial to provide evidence of protection used hepatitis B core antigen (HBcAg) as a powerful source of stimulating T-helper cells. Four calves were immunized with recombinant HBcAg with the SR1 region fused as a surface loop structure (HBcAg-SRl), and four control animals were immunized with native HBcAg (Boulter et al., 1995). Saponin was used as the adjuvant and five immunizations were given at monthly intervals. After sporozoite challenge, all the calves became infected, but the immunized animals showed a marked reduction in the number of schizonts and piroplasms, and also had significantly longer prepatent periods (length of time until schizonts were seen in lymph node smears) compared to the controls. Furthermore, very high titres of anti-SR1 antibodies (> 1 in 10 240) were apparent only 14 days after the first immunization, and sera collected between the third and fourth immunizations exhibited very strong sporozoite neutralizing ability (84% inhibition of sporozoite invasion into host cells when used at a dilution of 1 in 250). Strong CD4' T-cell responses were observed, although evidence of a T-suppressor element within the SR1 region was also noted. Importantly, all the test calves survived challenge whereas two of the control calves did not (Boulter et al., 1995). The subsequent trial utilized the full-length SPAG- 1 molecule, expressed with a his6 tag (an N-terminal run of six histidine residues) to facilitate purification, formulated with RWL@, a proprietary adjuvant from SmithKline Beecham, or incorporated into immunostimulatory complexes (ISCOMs) (Boulter et al., 1998, 1999). Six animals were immunized three times with SPAG-1-RWL and six animals with SPAG-1 in ISCOMs, whilst controls were immunized with phosphate-buffered saline (PBS) and adjuvant (PBS-RWL) or empty ISCOMs. A further group was immunized with p67-RWL, the SPAG-1 homologue in T. parva, to see if any crossspecies protection could be induced with a T. annulata challenge. On sporozoite challenge all animals developed classical symptoms of tropical theileriosis but of varying intensities. Briefly, the SPAG-1 -RWL group were the best protected as assessed by an increase in the prepatent and incubation periods, and the observation that three of six animals survived challenge whereas all 12 controls (six PBS-RWL and six empty ISCOMs)

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did not. Again, significant levels of anti-SPAG-1 antibodies, sporozoite neutralizing antibodies and T-cell proliferative responses were obtained, although there was no correlation between these and the level of protection induced (Boulter et a/., 1998, 1999). The SPAG-1-ISCOM immunized group exhibited very limited anti-SPAG- 1 antibodies and T-cell responses, but two did survive challenge. Interestingly, the p67-RWL group also showed some protection to T . annulata challenge as indicated by the fact that three of six survived. Significant levels of anti-SPAG-1 antibodies recognizing determinants in the extreme N- and C-termini and sporozoiteneutralizing antibodies were also produced by this group, indicating that there is some level of cross-species protection induced (see also Figure 2). The reciprocal trial, in which animals were immunized with SPAG-1RWL but then challenged with T . parva sporozoites, was subsequently conducted (N. Boulter et a/., unpublished data). Preliminary results indicated that, whilst all animals became infected, four of ten animals immunized with SPAG-1 -RWL were protected against a 70% lethal dose of T. parva, whereas four of seven animals immunized with p67, and only one in seven of the control group, were protected. The existence of cross-reactive epitopes between SPAG-1 and p67 is well documented (Knight et a/., 1996; Figure 2), but these studies raised the possibility of the development of a single vaccine that would be effective against both parasites. (c) Tams trial. In the trial with Tams, two allelic forms of the major merozoite surface antigen (Tamsl- 1 and Tams 1-2) were included. Tams 1- 1 and 1-2 proteins were produced. They were expressed in Escherichia coli with his6 tag. They were also expressed in Salmonella typhimurium aroA vaccine strain SL3261 (d’oliveira et al., 1996). Naked DNA constructs with Rous sarcoma virus long terminal repeats (LTRs) as the promoter were also prepared. Five groups of three cattle were used as follows: one group was immunized with his6 tagged Tams 1- 1/ 1-2 incorporated into ISCOMs; a second group was immunized with naked DNA plasmids encoding the antigens; a third and fourth group were immunized with recombinant S . typhimurium via the subcutaneous or oral routes, respectively; and the fifth group contained unimmunized controls. The immunizing regimen differed between the groups but all animals were challenged 4 weeks after the last immunization with a stabilate made from blood taken from an infected animal with 30% piroplasm parasitaemia (d’oliveira et a/., 1997). The Tams-ISCOM immunized group developed anti-Tamsl-1 and 1-2 antibodies by the time of challenge and were protected against blood challenge as designated by a parasitaemia of 1 % or less (compared to an average of 16% in the controls) and a maximum rectal temperature less than 41 “C. Two of the three DNA-immunized animals survived challenge despite the absence of detectable anti-Tams antibodies. In contrast, none of the Salmonella immunized animals was


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protected against challenge, and two of the animals died. All three controls died (d’oliveira et al., 1997). Whilst one must, of course, be cautious about the interpretation of such small-scale vaccine trials, the results provided some encouragement concerning the feasibility of developing a subunit vaccine against tropical theileriosis. Obviously, further work needs to be conducted in order to try to enhance the degree of protection by utilizing alternative delivery systems and adjuvants, and by including more antigens, particularly from the schizont stage.

3. THElLERlA PARVA 3.1. Overview

Animals recovered from a T. parva infection are solidly immune to a homologous lethal challenge (reviewed by Irvin and Morrison, 1987; Morrison and McKeever, 1998). However, similar animals usually succumb to heterologous challenge owing to antigenic polymorphism, a feature which distinguishes T. parva from T. annulata. Live vaccines based on the administration of oxytetracycline with a sporozoite stabilate (‘infection and treatment method’) produce a mild or subclinical infection and induce strong protection (see Sections 1.3.4, and 3.3.1; Radley, 1981; Irvin and Morrison, 1987; Dolan, 1989). Most research to date is consistent with the view that immunity induced by infection with T. parva is cell mediated (Figure 3). A body of evidence exists which implicates MHC-restricted cytotoxic T cells as important effectors in this immunity (Section 3.2.2, p. 69; Morrison and McKeever, 1998). However, other processes may well be important, and it is the purpose of this section to explore what is known and to speculate about what may be significant with respect to vaccine design. Immune responses to the sporozoite, whilst probably not important in the immunity induced by infection and treatment, can be significant. The strongest evidence to support this statement is the fact that protection against sporozoite needle challenge can be engendered in a proportion of animals by vaccinating with recombinant p67 protein, a sporozoite surface antigen (see Sections 3.3.3 and 3.3.4 p. 75, 76; Musoke et al., 1992). The basis for this immunity is not known but there is no correlation with neutralizing antibody titre, whilst antigen-reactive T cells are difficult to detect. Nevertheless, this is very encouraging and field trials to evaluate this antigen under natural challenge are being undertaken. A final subunit vaccine formulation will almost certainly require inclusion of a schizont component.

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Unknown anti-sporozoite effector induced by p67 vaccination

t

Trophozoite development 24 hours

CD4+

I

* Cytos atic rnacrophage

Inhibition by cyiostasis p

BOLA-restricted

Figure 3 Summary of the proposed immune response against Theileriu parvu. The figure is drawn to emphasize the proposed central role for BOLA-restricted CTLs directed against schizont-infected T cells. An important role for anti-sporozoite immunity (of unknown character) induced by immunization with recombinant p67 is also stressed. Potential roles for neutralizing anti-sporozoite antibodies as well as effectors of the innate immune system are suggested. Abbreviations as in Figure 1.


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3.2.1. Sporozoite Naive animals which have recovered from a single acute experimental infection with T . parva will not have significant serum antibodies to the sporozoite stage but they will be immune to homologous sporozoite challenge. The basis for this immunity is almost certainly a cell-mediated response to the schizont-infected lymphocyte (Figure 3). However, animals under persistent field challenge do acquire sporozoite neutralizing antibodies, as do animals hyperimmunized with sporozoite lysates (Musoke et al., 1982, 1984). One very important aspect of this neutralization activity is that it is largely directed at highly conserved epitopes (Musoke et al., 1984). These sera recognize a range of polypeptides, detected by immunoblotting, ranging in size from 150 to 24 kDa with one very prominent species of 67 kDa (Iams et al., 1990a). This antigen, dubbed p67, was also defined by several neutralizing monoclonal antibodies (Musoke et al., 1982, 1984, 1992; Dobbelaere et al., 1985a,b). As discussed later, this antigen in recombinant form is a prime vaccine candidate and induces complete or partial immunity in a proportion of animals. The only other fully characterized antigen from the sporozoite is a microneme/rhoptry antigen of 104 kDa (Iams et al., 1990b), but antibodies to this molecule do not neutralize sporozoite invasion in vitro. In addition, vaccination with this antigen does not induce any protection (S. Morzaria, personal communication). 3.2.2. Trophozoite and Schizont (a) The cell-mediated response - a perspective. As stated earlier, the main focus of protective immunity to T . parva is the schizont-infected cell (Morrison and McKeever, 1998). T . parva schizonts are thought to reside primarily in T cells in vivo (cf. T . annulata, which resides principally in macrophages/monocytes), although the sporozoite can invade different lymphocyte lineages in vitro (Baldwin et al., 1988; Glass et al., 1989; Spooner et al., 1989; Campbell et al., 1994; Morrison et al., 1996; Forsyth, 1997; Forsyth et al., 1997, 1999). The effector mechanism(s) against the schizont is (are) undoubtedly cellular. This assertion is based partly on the fact that serum from infected/immune animals, whilst containing anti-schizont antibodies, will not passively confer immunity on a naive recipient (Theiler, 1907; Muhammed et al., 1975). More importantly, T cells can, however, successfully transfer immunity from an immune twin to the non-immune identical partner (Emery et al., 1981; McKeever et al., 1994). All the available evidence is consistent with the main effector mechanism being a

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cytotoxic response mediated by MHC class 1-restricted CD8+ T cells (Figure 3; Morrison and McKeever, 1998). The first indication of this mechanism was provided by Pearson et al. (1979), who demonstrated that PBMs from immune cattle underwent a proliferative response to schizontinfected autologous cells - the so-called ‘autologous MLR’. Furthermore, they showed that the resulting population of primed cells contained cytotoxic activity against schizont-infected autologous targets and, interestingly, also some lesser activity against schizont-infected allogeneic cells. The detailed kinetics of the MHC-restricted response were first documented by Emery et al. (1981) and Eugui and Emery (1981), who showed that CTLs were transiently detectable in the peripheral blood leucocytes of calves recovering from an infection and treatment regimen for 2-3 days at the remission period. MHC-restricted CTLs also appear transiently in immune animals following challenge (Morrison et al., 1987). In contrast, calves undergoing a lethal reaction produce totally unrestricted cytotoxic responses late in the infection (Emery et al., 1981), which may underlie the massive destruction of lymphocytes that is a major pathological feature of this disease. The MHC-restricted CTLs are CD8+ and are highly specific, as they recognize only T. parva schizont-infected cells. Depending on the immunization protocol, these CTLs can be specific to a particular parasite strain (Morrison et al., 1987, 1995; Morrison and Goddeeris, 1990). Typing and blocking with monoclonal antibodies against class 1 MHC has confirmed that the response requires a minimum of one shared specificity between target and donor (Morrison et al., 1987). Very strong evidence that CD8+ cells have a role in vivo has been obtained by adoptive transfer. The population of cells obtained by draining a lymph node of an immune animal responding to challenge was highly enriched for the CD8+ cells by depleting the y6 T cells, B cells and CD4+ cells using specific monoclonal antibodies and complement. These were transferred to one member of pairs of naive twins which had been infected 1-3 days previously with a lethal sporozoite inoculum. This resulted in recovery of these animals, whereas the controls (the other member of each pair) succumbed. Whilst these data are compelling, it must be noted that the depletion left a substantial proportion of cells that were negative for CD4, CD8, B and y6 T markers, which cannot formally be discounted. However, this criticism is allayed to a large extent since one calf received cells which had also been depleted of CD8+ cells and this showed no protection (McKeever et al., 1994). Immune animals clearly retain memory in their CTL pool for schizont antigens, as demonstrated by the increase in precursor frequency from around 1 in 10 000 to 1 in 30 in the efferent lymph around day 6 after sporozoite challenge (Taracha et al., 1992, 1995a; McKeever et al., 1994). Morrison and McKeever (1998) reported that the frequency of around 1 in


71

10 000 CTL precursors persisted from 5-6 weeks after immunization in animals that were not challenged, but they did not say for how long they were monitored. The role of other effectors, such as components of the innate immune system like macrophages, may be important but they have been given relatively little attention. CD4+ cells also probably have a role to play, even if it is a subservient one, as helper cells to the CD8+ effectors (Baldwin et al., 1987; Brown, W.C. et al., 1989a,b, 1990). Grab et al. (1992) characterized a 24 kDa schizont antigen, which was specifically recognized by two CD4+ clones reactive with schizont-infected cells. The contribution of such clones could involve cytotoxic properties as well, since one report described a cloned CD4+ line with the ability to kill schizont-infected cells (Baldwin et al., 1992). However, since the bulk of the literature is about analysing the CD8+ CTL response, we shall concentrate on this theme. (b) The cell-mediated response - antigenic polymorphism. In crosschallenge experiments, frequently no protection is afforded in a proportion of animals, demonstrating that there is variation in the protective antigens (Radley et a/., 1975a,b). Immunizing with mixtures of as few as three isolates, however, led to wider cross-protection against several isolates, suggesting that the extent of the antigenic diversity may be limited (Radley et al., 197%). One important observation is that cross-immunity is not always reciprocal. This is exemplified by the Muguga and Marekibuni stocks of T . parva: Marekibuni invariably protects against both itself and Muguga, whereas Muguga protects only a proportion of animals challenged with Marekibuni (Irvin et al., 1983). By several independent criteria, including molecular profiling, the Marekibuni isolate is clearly a mixture of genotypes, whereas the Muguga isolate is homogeneous (Morrison, 1996). However, the Marekibuni stock has been cloned (Marekibuni 3219) and, when this is used as a vaccine, it protects only a proportion of calves against Muguga challenge (Taracha et al., 1995a). This type of observation suggests that, as well as the protective antigens exhibiting polymorphism, cross-reactive epitopes exist even on clones but that there is a host-determined selection in the response. In other words, although the Marekibuni clone contains both specific and cross-reactive epitopes, some cattle will ‘prefer’ the crossreactive epitopes to the exclusion of the specific ones and these will resist cross-challenge. However, the converse can also occur when the genotype of the cow selects the stock-specific epitope and is therefore unable to resist cross-challenge. This will be explored in detail below. The importance of the CTL response as a mediator of protection is supported by the fact that there is complete concordance between CTL parasite specificity and protection. Thus the Muguga-immunized calves that resist Marekibuni challenge have CTLs that kill Marekibuni-infected cells in an MHC-restricted manner, whereas those that succumb to challenge exhibit only Muguga-specific CTLs (Taracha et al., 1995a). This cross-reactivity is exhibited by individual T-cell

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clones, indicating that the phenomenon is due to shared epitopes and, furthermore, CTLs raised against Muguga will kill a Marekibuni clone that is otherwise genetically distinct, indicating that the phenomenon is not due to a Muguga-like genotype in the Marekibuni stock. (c) The cell-mediated response - MHC restriction, dominance and epitope competition. The phenomena described above suggest that different MHC class 1 elements select different parasite epitopes to present to the CTLs. If so, one could predict that there would be a correlation between MHC type and parasite specificity. To address this directly, Goddeeris et al. (1990) examined 3 1 CTL clones derived from four Muguga-immunized calves. They tested their specificity against a panel of Marekibuni-infected target clones that were half-matched at MHC class 1 locus. The restricting element of the clone determined the pattern of targets recognized and all CTLs restricted by the same MHC type had identical recognition patterns. Thus, epitope selection is dictated by the host MHC class 1 restriction element and hence the degree of cross-reactivity, all other things being equal, will be determined by the preference of the host MHC. A distinct phenomenon is that, in MHC heterozygous animals, which are the majority, there is a dominance hierarchy such that certain haplotypes act as restriction elements in preference to the allelic counterpart - a form of allelic exclusion (Morrison et al., 1987; Taracha et al., 1995b; Morrison, 1996). Moreover, a similar dominance is observed between class 1 molecules expressed by the same haplotypes (most haplotypes have two expressed class 1 genes). A specific example is afforded by the BOLA A10 and KN104 determinants: when they are both expressed on the same haplotype, the response to the Muguga strain of T . parva is always restricted by the KN104 element. (d) The cell-mediated response - a role for cytokines? Is the effector response solely due to direct action by CTL or do soluble mediators such as cytokines have an effect? Established schizont-infected cell lines are not inhibited by any cytokine tested to date - IL-1, IL-2, IL-4, IL-6, IL-10, IFN-a, IFN-y and TNF-a (Morrison and McKeever, 1998). In fact, TNF-a and IL-2 actually enhance the growth of established schizont-infected cell lines, whilst, interestingly, TNF-a, IFN-y, IFN-a, IL-1 and IL-2 inhibit the trophozoite (i.e. the preschizont, post-sporozoite) stage (De Martini and Baldwin, 1991; Preston et al., 1992a; Visser et al., 1995). Interestingly, using a multiplex polymerase chain reaction (PCR), McKeever et al. (1997) tested 11 parasitized lines for IL6, TNF-a, IL2Ra, IL-10, IL-lp, IFN-y, IL-2 and IL-4. No line produced IL-lp or IL-4, whilst all lines produced IL-2Ra and IL-10. Each line, however, produced a unique profile. The upregulation of IL-10 could have a detrimental effect, as this cytokine dampens down CD4' cells, which could explain why no CTL is present in naive cattle undergoing a lethal infection.


73

All things considered, proof of a direct role for cytokines in protective immunity to T. parva is largely lacking, although indirect effects such as effector cell recruitment cannot be ruled out and are indeed quite probable. (e) The cell-mediated response - immunopathology. Naive cattle infected with T. parva exhibit lymphadenopathy, i.e. the draining lymph node enlarges about fourfold. This enlargement takes only 1 or 2 days, and occurs after schizont-infected cells are first detectable, at just over a week after infection on average (Morrison et al., 1981). Analysis of the node and efferent lymph revealed 25% lymphoblasts, most of which were uninfected - indeed, parasitosis is of the order of only 1% at this time (Emery, 1981b). These cells have been reported to be predominantly a,O T cells with the profile CD8+, CD2-, CD3+, a phenotype rare in healthy cattle. T-cell receptor VP analysis indicates that these cells are polyclonal (F. Houston and W.I. Morrison, personal communication). These cells do not possess any measurable effector function. Additionally, it is not possible to detect CTLs at any stage in a primary infection (Emery et al., 1981; Taracha et al., 1992). It has been speculated that this anomalous T-cell proliferative response results in paracrine stimulation of growth and division of parasitized cells, thereby contributing to the pathology. This phenomenon may be similar in some respects to the aberrant T-cell proliferation observed in T. annulata (see Section 2.1.3(b), p. 59). Preliminary analysis demonstrated that these cells secrete IL-10 and that IL-10 potentiates growth in vitro (Morrison and McKeever, 1998). Since, as already mentioned, parasitized cells express IL-10 in their own right, IL-10 could be pivotal to the immunopathology, especially since it would also suppress the development of a Thl CTL response, which is required for immunity. An interesting contribution to the understanding of pathogenesis was provided by the work of Morrison et al. (1996). These authors infected CD8+, CD4+ and B cells with sporozoites in vitro and then inoculated the resulting schizont-transformed lines back into the cow from which they had been derived. Infected CD4+ and CD8+ cells produced severe pathology, whereas B cells caused mild self-limiting reactions. The basis for this interesting observation remains obscure.

3.3. Vaccination 3.3.1. Live Vaccines

-

The ‘Infection and Treatment’ Method

Early work (Lowe, 1933; Wilson, 1950; Barnett, 1957) suggested that the number of infectious particles (sporozoites) was proportional to the disease severity, and this was confirmed by Cunningham et al. (1974) and Radley et al. (1974), who performed titration experiments using ground tick

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suspensions containing sporozoites. Since those animals that recovered were solidly immune to homologous challenge, it was realized that the basis for a live vaccine was at hand. However, variability in clinical outcome was too great to allow the use of titrated stocks of sporozoites as vaccines in their own right, and tetracyclines were used to control the disease progression. Initially, these were short acting and had to be given continually to limit the infection (Neitz, 1953; Brocklesby and Bailey, 1965), but the method was improved by using modified longer acting tetracyclines. Thus, first, N-pyrrolidinomethyl tetracycline (Cunningham et al., 1973; Brown, C.G.D. et al., 1977) was introduced (given for 3 days from the time of infection) and then this ‘infection and treatment’ method was refined by Radley (1981), who used a single dose of oxytetracycline co-administered with the sporozoites. However, not all stocks can be controlled by this regime and some require administration of the drug for up to 6 days. Clinical breakthrough of the vaccinating strain(s) has been reported in Malawi (Mbogo et al., 1996) and Kenya (Mutugi et al., 1991). Animals vaccinated by infection and treatment can resist challenge with a 1000-fold lethal sporozoite dose and can remain immune without challenge for more than 3 years. Owing to antigenic polymorphism, the ‘field’ protection afforded by any one stock is limited and in practice combination ‘cocktail’ vaccines are used to give wider coverage. However, this is a controversial practice as there is concern that such vaccines actually enhance the spread of novel variants into the areas where they are used and, for this reason, the alternative practice of preparing local vaccines is recommended in some countries (Dolan, 1987). A very important point, worth re-emphasizing, is that it is not possible to produce an attenuated cell line vaccine against T. parva that is equivalent to those used so successfully against T. annulata. The principal reason for this is apparently the poor ability of the T. parva schizonts to transfer from donor host cells to recipient host cells. Indeed, the efficiency of T. parva transfer is at least two orders of magnitude lower than that achieved by T. annulata (see Irvin and Morrison, 1987). The reason for this difference is unknown but is probably connected with the difference between the host cells preferentially invaded by the two species. It is established that, in vivo, T. annulala resides predominantly in monocyte/macrophages, whilst T. parva is found largely in T cells (Morrison et al., 1996; Forsyth et al., 1997). Inoculation of a recipient with infected cells of either species will lead to an allogeneic graft rejection response, thus releasing the schizonts. Presumably this rejection and schizont release is equally efficient in the case of both T. parva and T. annulafa. Therefore, it is likely that the difference in transfer efficiency is at the level of entry into the new recipient host cells. As the recipient host cells of T. annulata are phagocytes (monocytes), whilst those of T. parva are not, a mechanism for enhanced transfer of T. annulata is


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provided. This argument, of course, assumes that the preferred host cell invaded by the transferring schizont is the same as that invaded by the sporozoite, which we doubt has been established. 3.3.2. Live Vaccines - Mild Strains of T. parva as Vaccines An alternative method to infection and treatment, which, as stated above, can lead to clinical disease, is the use of naturally occurring mild strains of T. parva (see Barnett and Brockelsby, 1966a; Koch et al., 1988; Mbogo et al., 1996). In their 1996 study, Mbogo et al. demonstrated that one mild T.parva strain (Lanet, NVRC stabilate 263), inoculated as a sporozoite stabilate, cross-protected against five virulent stocks of T . parva, including one buffalo-derived isolate. The protection against the four cattle-derived isolates was complete, whereas three of five animals challenged with buffalo isolates died but the other two were completely protected. These are promising results and this type of vaccine has real potential, although the possibility of reversion to virulence after tick passage, as was demonstrated by Barnett and Brockelsby (1966b), is a serious counter consideration. 3.3.3. Dead Vaccines

-

p67, a Recombinant Sporozoite Antigen

For many reasons, it would be desirable to replace the current live vaccines with non-living material. This would have the advantages of being noninfective, free of carrier pathogens and, hopefully, more stable. Attempts to induce protection using killed parasites or parasite extracts from other life-cycle stages of the parasite result in the production of serum antibodies but no protection is engendered. However, with the sporozoite stage, it is possible to induce neutralizing antibodies, and this observation in principle means that it should be possible to reduce the sporozoite inoculum and limit the infection. This line of thinking led to the search for sporozoite antigens with neutralizing determinants and a major candidate was defined using mAbs (Musoke et al., 1984; Dobbelaere et al., 1985a,b). This molecule, called p67, was shown to be recognized by immune bovine sera, highly conserved, and surface-located (see Section 3.2.1., p. 69; Dobbelaere et al., 1985a,b). The gene for this antigen has been cloned, and contains 709 codons and is homologous to the SPAG-1 gene of T. annulata, particularly at the N and C termini (Figure 2; Knight et al., 1996). Interestingly, the p67 and SPAG-1 antigens each contain the elastin motif PGVGV, although this occurs only once in p67 and 17 times in SPAG-1 (Nene et al., 1992). The two antigens have also been shown to cross-react immunologically, and sera and mAbs against the two antigens cross-neutralize (Figure 2).

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The p67 antigen has been expressed in a number of systems, including E. coli, baculovirus, vaccinia and Salmonella dublin (see Musoke et al., 1992; Nene et al., 1995; Heussler et al., 1998; Honda et al., 1998). In the E. coli system, the antigen has been used in vaccination trials when expressed as a C-terminal fusion to the non-structural protein 1 (NS1) of influenza virus and as a his6 tag product. The first published trial was using the NS1 fusion: nine indigenous Boran (BOSindicus) cattle were immunized five times with 3% saponin as the adjuvant (Musoke et al., 1992). Six of the animals were protected from a 70% lethal dose (LD70) homologous challenge with T.parva Muguga. Protection of four of the six was complete, showing no evidence of clinical reaction, whilst the other two underwent a mild reaction. The other three calves immunized with p67 had severe reactions, as did all ten controls. The mechanism of protective immunity was unclear. All the nine animals developed neutralizing antibodies but the titres did not correlate with protection. The authors reported that two of the non-reactors were PCR-negative using specific T. parva primers on lymph node biopsy material obtained 60 days after the challenge, suggesting that sterilizing immunity had been induced. An important follow-up investigation reproduced the initial results by observing protection in 7 of 12 calves (two of which were completely protected) against homologous challenge (Nene et al., 1996). More importantly, the authors reported that they protected 6 of 11 calves (one completely) against a heterologous challenge with a stock (T. parva Marekibuni) which is not cross-protective by infection and treatment. The same group of authors has evaluated a number of other expression and antigen delivery systems. Baculovirus-derived p67 was produced in an attempt to obtain ‘native’ antigen, but the material produced was unfortunately only weakly glycosylated and the bulk of it was produced as a series of partially processed forms, most of which failed to react with a mAb (TPM12) that recognizes native p67. None the less, four of six Boran cattle were protected by this material against homologous challenge (Nene et al., 1995). Recently, it was reported that, in trials using 86 cattle in total, 70% protection was obtained using p67 (Morrison and McKeever, 1998).

3.3.4. Recombinant Vaccines - Live Delivery Systems The protection induced by recombinant p67 relies on multiple vaccinations with antigen in adjuvant. Clearly this is not an ideal delivery strategy from a practical viewpoint and simpler systems would be preferable. In addition, it has proved difficult to detect significant T-cell responses using p67 formulated in adjuvant and lack of this type of immunity may be compromising the effectiveness of p67 as a protective immunogen. Hence two live delivery vehicles, Salmonella and vaccinia, have been evaluated in the context of p67.


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In the first trial, Heussler et af. (1998) immunized a group of seven calves intramuscularly on 3 monthly occasions with 2 x lo9 colony-forming units of S. dubfin, composed of an equal mixture of three populations of expression transformants. These consisted of high, medium and low copy number expression plasmids; in addition, the high copy plasmid exported the antigen to the periplasmic space, whilst the others retained it in the cytoplasm. Controls consisted of seven naive calves and seven calves immunized with S . dubfin alone. Six of the experimental animals underwent mild or moderate reactions on challenge, whilst one animal was completely protected. The controls were less protected, with two calves in each group experiencing severe theileriosis. Statistically, the differences were significant and indicated protection. In a follow-up study, Gentschev e f af. (1998) attempted to compare the effect of cytoplasmically located p67 with secreted p67 using S. dubfin as the live host. Oral and intramuscular routes of delivery were also assessed. Again, multiple inoculations were used. The secreted p67 was made by attaching the C-terminal E. coli haemolysin secretion signal sequence. The results indicated that secreted antigen engendered better protection than the internally located antigen and that intramuscular delivery was marginally superior to the oral route. However, the results must be regarded as preliminary owing to the small numbers (three) of animals per group. Recombinant p67 delivered via vaccinia virus has been evaluated by Honda et af. (1998). Again, this system is capable of inducing T-cell responses, particularly CD8+ cells. The p67 vaccinia construct given alone to seven animals (two inoculations) did not have any protective effect. However, when given in conjunction with a vaccinia construct engendering production of IL2, five of seven calves were protected. A co-administered vaccinia construct stimulating IL-4 enhanced production of antibody to p67 but had no effect on protection. T h s live delivery approach has much potential merit, particularly if the system could be reduced to a one-shot application. The antigen p67 is a very remarkable molecule, as it was unexpected that a single sporozoite component would induce significant protection on its own. Also, its partially protective effect against T. annulata is intriguing (Boulter et al., 1999). Research to elucidate the mechanism of immunity in completely protected animals should be a priority.

4. THElLERlA SERGENTI 4.1. Classification

Taxonomically, there is a debate about the classification of the relatively mild species of Theileria that constitute what can be loosely described as the

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T. buflelilorientalislsergenti group (Fujisaki et al., 1994; and see Section 1.1.1, p. 43). These organisms (or this organism) are (is) characterized by being relatively non-pathogenic and inducing symptoms associated with the piroplasm stage of the life cycle. One of the defining characteristics is the rarity of the schizonts, which occur in markedly enlarged host cells, and have been observed in the draining lymph node, liver and spleen and, sometimes, extracellularly (Minami et al., 1990; Kawazu et al., 1991; Sat0 et al., 1993, 1994). For the purposes of this review, with its focus on immunity, we shall use the term T. sergenti to describe the organism(s) that infect cattle in Japan and Korea, where they cause chronic anaemia. The Japanese and Korean parasites are transmitted by the tick Haemaphysalis longicornis. 4.2. Clinical Features and Control

As stated above, the major pathological phase of the life cycle of T. sergenti is the piroplasm stage, which leads to severe chronic anaemia, particularly in susceptible ‘western’ breeds of cattle such as Holsteins. Indigenous breeds such as native Korean cattle and Japanese Black calves are relatively resistant (Baek et al., 1992a; Terada et al., 1995). One interesting feature of the disease is that a carrier state is established, with the consequence that stress-induced relapses occur (Sugimoto, 1997). Recovery from infection induces immunity to homologous challenge and a live blood vaccine has been used as a control measure, but is not now recommended. The major method of control is using anti-theilerial drugs.


Analyses of the immune response in T. sergenti-infected calves are very limited (see Figure 4). There is a humoral response, first described by Takahashi et al. (1972), who used an indirect fluorescent antibody (IFA) assay to demonstrate antibodies to the piroplasm by day 5, which reached a peak on day 20 and persisted until day 60. Convalescent sera reacted with piroplasm antigens of 23, 29, 32 and 67 kDa in Western blots (Ohgitani et al., 1987). mAbs directed against the 32 kDa polypeptide (p32) (Kobayashi et al., 1987) are able passively to confer resistance against merozoite challenge (Tanaka et al., 1989). Protection against challenge is afforded by vaccination with synthetic p32, as described in more detail below. Cell-mediated immunity is also detectable in calves undergoing a clinical infection. Yasutomi et al. (1991) observed T-cell proliferation of PBMs directed against merozoites in infected blood. The peak of proliferation occurred about 3 weeks after infection and the response was ablated by

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Sporozoites

Neutralizing antibodies

Innate immunity

Cell-mediated immunity e.g. CTL

. macrophage?

e.g. anti-p32/33 Piroplasm

+ Figure 4 Summary of the proposed immune response against Theileria sergenri. This diagram highlights a role for neutralizing antibodies directed at the merozoite/piroplasm stages. The other suggested effectors are speculative and clearly indicate our general state of ignorance of the immune mechanisms involved in modulating T . sergenti infections. Abbreviations as in Figure 1.

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anti-T cell mAbs and by autologous infection serum. However, interestingly, stimulation of proliferation resulted from inclusion of a mAB against merozoites. The same authors detected cytotoxic cells, reaching a peak at weeks 7-8 after infection, which killed a bovine leukaemia cell line and were probably NK effectors. Co-culture with merozoites caused a slight enhancement of the degree of cytotoxicity, whereas further addition of autologous serum (taken at weeks 5-6 after infection) caused a marked reduction. Asaoka et al. (1991) conducted a detailed study of macrophage activation in calves infected with T. sergenti. Specifically, they studied the oxidative burst induced either specifically by merozoites or non-specifically by zymosan. Six calves produced activated macrophages that responded, for up to one month after inoculation, to merozoites, particularly those that were opsonized with homologous or heterologous antisera. However, the parasites were not eliminated at the time of the peak oxidative burst in vivo, suggesting that this mechanism may not be fully effective. On the other hand, Ishii and co-workers (1992) demonstrated that the inhibition of activated monocytes in vivo by prednisolone correlated with an increase in parasitaemia, suggesting an important effector role for these cells.

4.4. Vaccination with Non-living Components

Vaccination with non-living components has been reported by two groups in Korea and Japan (Baek et al., 1992a,b, 1994; Onuma et al., 1997). Baek and co-workers in Korea prepared crude soluble extracts by sonication of merozoites and centrifugation at 20 000 g, and then used this material to vaccinate calves. Freund’s complete adjuvant was used in the primary injection and Freund’s incomplete adjuvant in the booster dose 4 weeks later; 100 mg of crude antigen were given per animal per injection. Nine weeks after the boost, calves were challenged with 5.6 x lo6 erythrocytes at 40% parasitaemia with the homologous parasite. The vaccinated animals and the controls both developed parasitaemia, but the vaccinated did not become anaemic, whereas the controls did. The serum of vaccinated animals predominantly recognized the 33 kDa antigen. The same authors inoculated 20 Holstein calves and placed them under heavy natural tick challenge together with 20 control calves. All 20 controls needed chemotherapy (five requiring blood transfusions), whereas only six of those vaccinated required treatment. It is probable that the effect was mediated primarily by an immune response directed to the dominant 33 kDa antigen. The 33 kDa antigen is thus a candidate for a subunit vaccine. The gene was therefore cloned and shown to be a highly polymorphic system (Kawazu et al., 1991; Matsuba et al., 1993a,b). Recombinant antigen has been produced in the baculovirus system and synthetic multiple antigenic peptides


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(MAPS)have been made containing a KEK motif. Animals were vaccinated four or five times with these antigens, using a range of doses and Freund’s complete adjuvant or liposomes as adjuvants. Protection against both parasitaemia and clinical symptoms was achieved (Onuma et al., 1997). The prospects for producing a recombinant vaccine against T. sergenti, where a strong humoral anti-merozoite response is probably all that is required, must be better than those for either of the other two species considered.

5. COMPARATIVE ASPECTS

All three species have much in common biologically. However, it is the differences that are perhaps worth stressing (Table 1). From an immunological point of view, the differences are marked. To begin with, it is possible to immunize against T. annulata with attenuated schizont-infected cells, whereas this is not possible with T . parva (because the efficiency of parasite transfer is too low) or T. sergenti (because the schizont stage is too rare). The influence of the host cell type in which the schizont resides probably underlies the differential ability of T. annulata to transfer at a 100 times greater frequency than T. parva. Specifically, as T. annulata inhabits a phagocyte (the macrophage), it is provided with an efficient process for schizont uptake after the donor host cell has been destroyed by allograft rejection. T.parva, residing in T cells that are non-phagocytic, is not availed of the same process. The immunity induced against the schizont-infected cell is known to be cell mediated in T. parva and T. annulata infection, but no data exist for T. sergenti (Figures 1 , 3 and 4). With T. parva the main effector is the MHCrestricted CTL, whereas for T. unnulata the most important effector is the macrophage with a secondary role for CTLs. The secreted products of macrophages, such as NO and TNF-a, are thought to be important in protective immunity to T. annulata. The attenuated T. annulata vaccine is claimed to have efficacies of more than 90% and is cross-protective against different field isolates. The infection and treatment regime for T. parva protects against homologous challenge only. Presumably this is because T. parva exhibits polymorphism of the target antigens recognized by the CTLs. The CTL response against T. parva exhbits complex phenomena, such as epitope competition and dominance of certain BOLArestriction elements. NK cells probably play a role in T. parva and T. annulata infections. Passive immunization with a mAb against the major merozoite antigen indicates a key role for antibody in immunity to T. sergenti. This is largely due to the fact that the only pathogenic phase of the life cycle is the piroplasm. This compares with T. annulata in which pathogenesis is associated with both the schizont

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and piroplasm phases, and contrasts with T. parva infection, in which the pathology is solely a function of the schizont stage. Both T. parva and T. annuluta induce polyclonal autologous T-cell proliferation. In the case of T. annulata, this is thought to damage the normal immune response by effectively depleting and/or overwhelming the antigen-specific T-cell pool. Vaccination with the major sporozoite surface antigen p67 gives sterilizing immunity against T. parva sporozoite challenge in a proportion of animals and partial immunity in others, whilst some are fully susceptible. The immunological and genetic bases for this phenomenon are not understood, but it is not observed with T. annulata when immunizing with the homologous antigen SPAG-1 (with which only partial immunity is obtained). The major merozoite antigen (30-33 kDa) provides protection against T. sergenti and also against a blood challenge with T. annulata. It is considered unlikely that this component will be needed or effective against T. parva, in which the piroplasm plays a secondary role.

6. THE FUTURE

Future immunological and vaccine research in bovine theilerioses must be aimed at usable, effective products within the next decade. This is a tall order, especially if we demand certain standards, such as that a T. annulaiu vaccine must be as effective as the current attenuated ones. It is highly likely that the vaccines that emerge will be of the ‘naked’ DNA type and will include selective immunopotentiators in the form of cytokine genes. The focus of attention for T. sergenti is likely to remain surface proteins of the merozoite, although, if the methodology became available, schizont constituents would be useful components also as, in principle, merogony could be inhibited. Of paramount importance is the need to define protective schizont antigens for T. parva and T. annuluta, which we think are going to be fundamental in the production of effective vaccines. For T. parva and T. annuluta, multistage, multicomponent vaccines are likely to be preferred, but this may create difficulties about whether the required spectra of immune responses are fundamentally compatible. Much basic immunological research and empirical vaccine testing lies ahead.

ACKNOWLEDGEMENTS We thank Drs Rachel Adamson, John Campbell, Ivan Morrison and Pat Preston for critically reading this manuscript. Thanks also to Duncan


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Brown and his team for all their support over the years. We wish Duncan a happy retirement. Original work referred to from the authors’ laboratory was funded by the Biotechnology and Biological Sciences Research Council, the European Union (contract numbers CT95 0003 and CT91 0019) and the Wellcome Trust (grant numbers 0312219 and 040179).

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Preston, P.M., Brown, C.G.D., Bell-Sakyi, L., Richardson, W. and Sanderson, A. (1992b). Tropical theileriosis in Bos taurus and Bos taurus cross Bos indicus calves: response to infection with graded doses of sporozoites of Theileria annulata. Research in Veterinary Science 53, 230-243. Preston, P.M., Brown, C.G.D., Entrican, G., Richardson, W. and Boid, R. (1993). Synthesis of tumour necrosis factor-alpha and interferons by mononuclear cells from Theileria annulata-infected cattle. Parasite Immunology 15, 525-534. Preston, P.M., Jackson, L.A., Sutherland, LA., Bell-Sakyi, L., Wilkie, G., Brown, D.J., Schofield, J., Melrose, T.R., Sanderson, A. and Brown, C.G.D. (1998). Theileria annulata: the expression of two novel macroschizont antigens on the surface of infected mononuclear cells differs during in vitro attenuation of a virulent cell line. Experimental Parasitology 89, 228-240. Preston, P.M., Hall, F.R., Glass, E.J., Campbell, J.D., Darghouth, M., Ahmed, J., Spooner, R.L., Jongejan, F., Shiels, B.R. and Brown, C.G.D. (1999). Theileria annulata: innate and adaptive immune responses cooperative in protective immunity. Parasitology Today 15, 268-274. Purnell, R.E. and Chang Rae, M. (1981). The use of imidocarb diproprionate for the treatment of Theileria sergenti infections of cattle. Australian Veterinary Journal 51, 224-227. Radley, D.E. (1981). Infection and treatment method of immunization against theileriosis. In: Advances in the Control of Theileriosis (A.D. Irvin, M.P. Cunningham and A.S. Young, eds), pp. 227-237. The Hague, Boston, London: Martinus Nijhoff Publishers. Radley, D.E., Brown, C.G.D., Burridge, M.J., Cunningham, M.P., Pierce, M.A. and Purnell, R.E. (1974). East Coast fever: quantitative studies of Theileria parva in cattle. Experimental Parasitology 36, 278. Radley, D.E., Brown, C.G.D., Burridge, M.J., Cunningham, M.P., Kirimi, I.M., Purnell, R.E. and Young, A.S. (1975a). East Coast fever: I . Chemoprophylactic immunization of cattle against Theileria parva (Muguga) and five theileria strains. Veterinary Parasitology 1, 35-41. Radley, D.E., Young, A.S., Brown, C.G.D., Burridge, M.J., Cunningham, M.P., Musisi, F.L. and Purnell, R.E. (1975b). East Coast fever. 2. Cross-immunity trials with a Kenya strain of Theileria lawrencei. Veterinary Parasitology 1, 43-50. Radley, D.E., Brown, C.G.D., Cunningham, M.P., Kimber, C.D., Musisi, F.L., Payne, R.C., Purnell, R.E., Stagg, S.M. and Young, A S . (1975~).East Coast fever. 3. Chemoprophylactic immunization of cattle using oxytetracycline and a combination of Theileria strains. Veterinary Parasitology 1, 5 1-60. Rehbein, G., Ahmed, J.S., Zweygarth, E., Schein, E. and Horchner, F. (1981a). Immunological aspects of Theileria annulata infection in cattle. 1. E, EA, and EAC rosette forming cells in calves infected with T. annulata. Tropenmedizin und Parasitologie 32, 101- 104. Rehbein, G., Ahmed, J.S., Schein, E., Horchner, F. and Zweygarth, E. (1981b). Immunological aspects of Theileriu annulata infection in cattle. 2. Production of macrophage migration inhibition factor (MIF) by sensitized lymphocytes from Theileria annulata-infected calves. Tropenmedizin und Parasitologie 32, 154156. Richardson, J.O., Forsyth, L.M.G., Brown, C.G.D. and Preston, P.M. (1998). Nitric oxide causes the macroschizonts of Theileria annulata to disappear and host cells to become apoptotic. Veterinary Research Communications 22, 3 1-45. Robinson, P.M. (1982). Theileria annulata and its transmission - a review. Tropical Animal Health and Production 14, 3- 12.

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Sato, M., Kamio, T., Kawazu, S., Taniguchi, T., Minami, T. and Fujisaki, K. (1993). Histological observations on the schizonts in cattle infected with Japanese Theileria sergenti. Japanese Journal of Veterinary Science 55, 57 1 - 574. Sato, M., Kamio, T., Tanaka, S., Taniguchi, T. and Fujisaki, K. (1994). Development of Theileria sergenti schizonts in the lymph node of experimentally infected cattle. Japanese Journal of Veterinary Science 56, 715-722. Sayin, F., Dincer, S., Cakmak, A., Inci, A., Yukari, B.A., Vatansaerver, S., Nalbantoglu, S. and Deniz, A. (1997). Tick-borne disease in Turkey. Tropical Animal Health and Production 29 (suppl.), 53s. Scott, P. and Trinchieri, G. (1995). The role of natural killer cells in host-parasite interactions. Current Opinion in Immunology 7, 34-40. Sergent, E., Donatien, A., Parrot, L. and Lestoquard, F. (1945). Etudes sur les Piroplasmoses Bovines. Algiers: Institut Pasteur d’AlgCrie. Shiels, B.R., McDougall, C., Tait, A. and Brown, C.G.D. (1986). Antigenic diversity of Theileria annulata macroschizonts. Veterinary Parasitology 21, 1- 10. Shiels, B., Hall, R., Glascodine, J., McDougall, C., Harrison, C., Taracha, E., Brown, D. and Tait, A. (1989). Characterization of surface polypeptides on different life-cycle stages of Theileria annulata. Molecular and Biochemical Parasitology 34,209-220. Shiels, B.R., Smyth, A,, Dickson, J., McKellar, S., Tetley, L., Fujisaki, K., Hutchinson, B. and Kinnaird, J.H. (1994). A stoichiometric model of stage differentiation in the protozoan parasite Theileria annulata. Molecular Cell DlfSerentiation 2, 101-125. Shiels, B.R., d’oliveira, C., McKellar, S., Ben-Miled, L., Kawazu, S. and Hide, G. (1995). Selection of diversity at putative glycosylation sites in the immunodominant merozoite/piroplasm surface antigen of Theileria parasites. Molecular and Biochemical Parasitology 72, 149- 162. Shimizu, S., Yagi, Y., Nakamura, Y., Shimura, K., Fujisaki, K., Onodera, T., Minami, T. and Ito, S. (1990). Clinico-haematological observation of calves infected with Theileria sergenti. Japanese Journal of Veterinary Science 52, 13371339. Shirong, S. (1997). Application and effect of the schizont vaccine against Theileria annulata in China. Tropical Animal Health and Production 29 (suppl.), 101s102s. Singh, D.K. (1990). Methods currently used for the control of Theileria annulata: their validity and proposals for future control strategies. Parassitologia 32, 3340. Singh, D.K., Varshney, B.C., Raghav, P.R.S. and Thakur, M. (1993). Response of crossbred calves to immunization with Theileria annulata schizont infected lymphoid cell cultures. Indian Veterinary Journal 70, 605-608. Somerville, R.P.T. (1997). Molecular and Biochemical Characterisation of Nonattenuated and Attenuated Isolates of Theileria annulata. D.Phil. thesis, University of York. Somerville, R.P.T., Adamson, R.E., Brown, C.G.D. and Hall, F.R. (1998a). Metastasis of Theileria annulata macroschizont-infected cells in scid mice is mediated by matrix metalloproteinases. Parasitology 116, 223-228. Somerville, R.P.T., Littlebury, P., Pipano, E., Brown, C.G.D., Shkap, V., Adamson, R.E., Oliver, R.A., Glass, E.J. and Hall, F.R. (1998b). Phenotypic and genotypic alterations associated with the attenuation of a Theileria annulata vaccine cell line from Turkey. Vaccine 16, 569-575. Spooner, R.L., Innes, E.A., Glass, E.J. and Brown, C.G.D. (1989). Theileria annulata


95

and T . parva infect and transform different bovine mononuclear cells. Immunology 66, 284-288. Srivastava, A.K. and Sharma, D.N. (1981). Studies on the occurrence, clinical features and clinicopathological and pathomorphological aspects of theileriasis in calves. Veterinary Research Journal 4, 22-29. Stewart, N.P., de Vos, A.J. and Shiels, I. (1990a). Elimination of Theileria buffeli infections from cattle by concurrent treatment with primaquine phosphate and halofuginone lactate. Tropical Animal Health and Production 22, 109- 11 5. Stewart, N.P., de Vos, A.J., McHardy, N. and Standfast, N.F. (1990b). Elimination of Theileria buffeli infections from cattle by concurrent treatment with buparvaquone and primaquine phosphate. Tropical Animal Health and Production 22, 116-122. Stewart, N.P., Uilenberg, G. and de Vos, A.J. (1996). Review of Australian species of Theileria, with special reference to Theileria buffeli of cattle. Tropical Animal Health and Production 28, 81 -90. Sugimoto, C. (1997). Economic importance of theileriosis in Japan. Tropical Animal Health and Production 29 (suppl.), 49s. Sugimoto, C., Kawazu, S., Kamio, T. and Fujisaki, K. (1991). Protein analysis of Theileria sergentilbuffelilorientalis piroplasms by two-dimensional polyacrylamide gel electrophoresis. Parasitology 102, 341 -346. Sugimoto, C., Kawazu, S., Sato, M., Kamio, T. and Fujisaki, K. (1992). Preliminary biochemical characterization of ‘veil’ structure purified from Theileria sergenti-, T. buffeli- and T . orientalis-infected bovine erythrocytes. Parasitology 104, 207-213. Sutherland, I.A., Shiels, B.R., Jackson, L.A., Brown, D.J., Brown, C.G.D. and Preston, P.M. (1996). Theileria annulata: altered gene expression and clonal selection during continuous in vitro culture. Experimental Parasitology 83, 125133. Tait, A. and Hall, F.R. (1990). Theileria annulata: control measures, diagnosis and the potential use of subunit vaccines. Revue Scientifique et Technique, OfJice International des Epizooties 9, 387-403. Takahashi, K., Yamashita, S . , Isayama, Y. and Shimizu, Y. (1972). Serological response to the indirect fluorescent antibody test of cattle infected with Theileria sergenti. British Veterinary Journal 132, 112- 117. Tanaka, M., Ohgitani, T., Okabe, T., Kawamoto, S., Takahashi, K., Onuma, M., Kawakami, Y. and Sasaki, N. (1989). Protective effect against erythrocytic merozoites of Theileria sergenti infection in calves by passive transfer of monoclonal antibody. Japanese Journal of Veterinary Science 52, 63 1-633. Tanaka, M., Onoe, S., Matsuba, T., Katayama, S . , Yamanaka, M., Yonemichi, H., Hiramatsu, K., Baek, B., Sugimoto, C. and Onuma, M. (1993). Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA. Journal of Clinical Microbiology 31, 2565-2569. Taracha, E.L.N., Goddeeris, B.M., Scott, J.R. and Morrison, W.I. (1992). Standardization of a technique for analysing the frequency of parasite-specific cytotoxic T lymphocyte precursors in cattle immunized with Theileria parva. Parasite Immunology 14, 143- 154. Taracha, E.L.N., Goddeeris, B.M., Morzaria, S.P. and Morrison, W.I. (1995a). Parasite strain specificity of precursor cytotoxic T cells in individual animals correlates with cross-protection in cattle challenged with Theileria parva. Infection and Immunity 63, 1258-1262. Taracha, E.L.N., Goddeeris, B.M., Teale, A.J., Kemp, S.J. and Morrison, W.I.

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Theileria annulata infection), Ehrlichiu and tick toxicosis: present situation and proposals for future control strategies. Parassitologia 32, 41 -54. Young, A S . , Groocock, C.M. and Kariuki, D.P. (1988). Integrated control of ticks and tick-borne diseases of cattle in Africa. Parasitology 96, 403-432. Zablotsky, V.T. (1991). Specific prevention of bovine theileriosis in Soviet Union. In: Proceedings of the Second EEC Workshop on Orientation and Co-ordination of Research on Tropical Theileriosis (D.K. Singh and B.C. Varshney, eds), pp. 9-10. Anand, India: National Dairy Development Board.


The Distribution of Schistosoma bovis Sonsino. 1876 in Relation to Intermediate Host Mollusc-Parasite Relationships Helene Mone. Gabriel Mouahid and Serge Morand

Laboratoire de Biologie Animale. U M R n"55.55 du CNRS. Centre de Biologie et d'kcologie tropicale et me'diterrankenne. Universitk. Avenue de Villeneuve. 66860 Perpignan Cedex. France Abstract ..................................................................... 100 1. Introduction ............................................................. 100 102 2. Collection of Data ........................................................ 3. The Natural Mollusc Intermediate Host Spectrum .......................... 113 4 Geographical Distributions of the Mollusc Intermediate Hosts...............113 124 5. Geographical Distribution of S. bovis ...................................... 6. The Experimental Mollusc Intermediate Host Spectrum ..................... 125 7. Compatibility in the Mollusc-S . bovis Association ......................... 125 8 Three Main Populations of S bovis ....................................... 128 9 Paleobiogeographical Scenario of S. bovis................................. 130 10. Conclusion .............................................................. 132 Acknowledgements .......................................................... 133 References................................................................... 133

. . .

ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

.

Copyright Q 2000 Academic Press A / / rights o/reproducrion in any form reserved

100

ti. MONE ETAL.

ABSTRACT Schistosoma bovis is a digenean platyhelminth that is responsible for a parasitic disease called schistosomiasis or bilharziasis in bovines. It has a natural wide mollusc intermediate host spectrum and is compatible, experimentally, with a wide range of species. Our working hypothesis is that the Mediterranean Sea and the Sahara were two physical barriers that could have separated the populations of S. bovis in three parts and may have played a role in gene flow. Experimental data were collected from earlier published studies, and the different intermediate host spectra and the mollusc-parasite geographical compatibilities were compared between the North Mediterranean zone, the South Mediterranean zone and the South Saharan zone. From our results, the three major groups of S. bovis populations that could be determined were the Iberian, the Mediterranean and the South Saharan populations. Our tested hypothesis was thus not confirmed concerning the Mediterranean sea barrier but was confirmed with the Saharan one. A paleogeographical scenario of S. bovis is proposed following three major steps from a South Saharan origin to a possible local adaptation of the parasite in the Iberian Peninsula.

1. INTRODUCTION

Different species of animals coexisting in the same environment exert on each other reciprocal influences which can lead to coevolutionary processes (Futuyama and Slatkin, 1983). Parasites, as any other animals, are also under the selective pressures of the environment. The peculiarity of animal parasites is that they are closely associated with one or several species of animals called hosts. The host-parasite associations will coevolve under both the influences of the parasite on its host and of the host on the parasite (Frank, 1991). In addition to the morpho-anatomical, biochemical or behavioural markers, which may reveal an interpopulational genetic diversity, both host spectrum and host-parasite compatibility constitute useful markers as regards the knowledge of the transmission foci and of the risks of extension of the parasitism. The intermediate mollusc host spectrum corresponds to the number of different species of hosts a parasite may infect. This may range from a reduced host spectrum, where the parasite develops in only one species of host, to a wide host spectrum, where the parasite develops in many species of hosts.

DISTRIBUTION OF SCHlSTOSOMA BOVlS

101

The host-parasite compatibility can be defined as the result of both host susceptibility and parasite infectivity (Combes, 1985, 1995), and may show considerable variation among the population host-population parasite systems. It is genetically determined (Vkra et al., 1990; Richards et al., 1992) and constitutes the expression of the genetic variability of the host confronted to the genetic variability of the parasite. The host-parasite compatibility corresponds to the success of infection. It is usually expressed as the number of molluscs infected divided by the number of molluscs exposed to the miracidia (prevalence). This will be different depending on the geographical origin of both the parasite and the host, and on the species of host the parasite will use (Vkra et al., 1990; Ebert, 1994; Gandon et al., 1996; Morand et al., 1996). It will express the quality of the relationship existing between the parasite and its host. Schistosoma bovis Sonsino, 1876 (Platyhelminth, Digenean) was used as the parasite model. This parasite is the agent of a bovine debilitating disease called schstosomiasis or bilharziasis. It has a complex life cycle including two obligatory hosts: a vertebrate definitive host, and a mollusc intermediate host. Some authors suggest that the digeneans are primitive parasites of molluscs (Gibson, 1987; Rohde, 1994), whereas others suggest they are primitive parasites of vertebrates (Brooks and McLennan, 1991). It is generally agreed that the digenean-mollusc association is an ancient one. This association extends for at least 400 million years (Gibson, 1987). A synthesis of the biology and the ecology of S. bovis transmission, especially the patterns of the mollusc-parasite association, has been published recently (Mouahid, 1994). The geographical distribution of S. bovis is wide and covers different geographical zones, which are isolated by two major horizontal natural barriers: the Mediterranean Sea, and the Saharan Desert. These two barriers, even though not impassable, delimit the North-South geographical distribution of S. bovis in three major zones that we term the North Mediterranean zone, the South Mediterranean zone and the South Saharan zone, respectively. In other respects S. bovis presents large differences in both host spectrum and host-parasite compatibilities according to the geographical origins of both the host and the parasite. We took into account these peculiarities of S. bovis to analyse the different host-parasite geographical intermediate host spectrum and compatibilities in these three geographical zones. Our working hypothesis was that the natural barriers (Mediterranean Sea and Saharan Desert) have played a role in the gene flow between the different S. bovis populations. As no molecular data are available at the intraspecific interpopulational level of S. bovis, and as the host-parasite compatibility is the expression of the genetic relationship between the host and the parasite, we assumed that the data concerning the mollusc-S. bovis compatibility reflect the host-parasite coevolution in such a way as to propose a scenario of the biogeographical history of S. bovis.

102

H. MONE ETAL.

2. COLLECTION OF DATA

Data concerning the natural S. bovis-mollusc associations came from 45 published studies (Table 1). Data concerning the experimental S. bovismollusc associations were collected from 20 papers (Tables 2-6). The experimental results obtained using multimiracidial infections were considered. The number of miracidia used per snail varied from 2 to 100 but 83% of the prevalences concerned infections using 5 miracidia per snail. We made sure that the numbers of miracidia used for the multimiracidial infection tests were not correlated to the numbers of infected molluscs; the available data set (260 observations) showed such a bias did not exist (v = 29.85 + 0 . 3 7 ~ ; r = 0.09; 95% confidence interval - 0.03 and 0.21; P > 0.05). For each host-parasite association, the following parameters were checked: the species of mollusc; its geographical origin; the geographical origin of the schistosome; when possible, the number of miracidia used per mollusc during the exposure, the number of surviving or exposed molluscs which have been used for the infection test; and the number of infected molluscs on the number of surviving or exposed molluscs ( = prevalence, expressed in percentage). Mean prevalences were used when several data were recorded from the same population host-population parasite association. The albino populations of molluscs were considered as different populations from non-albino ones. All the prevalences were then expressed in proportions (p) and arcsine transformed (Zar, 1984). The ANOVA-factorial test together with the Scheffe’s F post-hoc Table I Bibliographic data on the natural geographical distribution Schistosoma hovis. Geographical zone

Country

Authors

of

Year

North Mediterranean zone

France (Corsica) Italy (Sardinia) Italy (Sicily) Spain

Brumpt Deiana Grassi and Ravelli Ramajo-Martin

1930 1954 1898 1972

South Mediterranean zone

Egypt Iraq

Sonsino MacHattie and Chadwick Al-Barrak et al. Arfaa et al. Lengy Blanc and Desportes

1876 1932

Iraq Iran Israel Morocco

1977 1965 1962 1936 (continued)

103

DISTRIBUTION OF SCHlSTOSOMA BOWS

Table I Continued. Geographical zone

Country Morocco Morocco Tunisia

South Saharan zone

Burkina Faso Cameroon Central African Republic Chad Congo Ethiopia Ethiopia The Gambia Ghana Guinea Guinea-Bissau Kenya Kenya Kenya Kenya Kenya Mali Mali Mauritania Niger Nigeria Nigeria Rwanda Senegal Somalia Sudan Sudan Sudan Tanzania Tanzania Tanzania Tanzania Tanzania Togo Uganda Zaire Zaire

Authors Dazo and Biles (cited by Southgate and Knowles) Freton et al. Anderson and Gobert Gillet (cited by Pitch ford) Ngonseu et al. Ngendaha yo Ngendahayo Fain and Lagrange Graber and Daynes Lo and Lemma Smithers Edwards and Wilson Gillet (cited by Pitchford) Gillet (cited by Pitchford) Southgate and Knowles Jelnes Ouma and Waithaka Southgate et al. Southgate et al. Ngendahayo Rollinson et al. Marill Vtra Cowper Ndifon et al. Wery (cited by Pitchford) Diaw and Vassiliades Sobrero Malek Bushara et al. Majid et al. Kinoti Dinnik and Dinnik Southgate et al. Kassuku et al. M wambungu Dogba Berrie Schwetz Chartier et al.

Year

1975a 1989 1934 1977 1991 1989 1989 1952 1974 1975 1956 1958 1977 1977 1975b 1983 1984 1985a 1989 1989 1990 1961 1991 1963 1988 1977 1987 1965 1969 1978 1980 1964b 1965 1980 1986 1988 1976 1964 1955 1990

A

0 P

Table 2 Snail infection experiments in PIanorbarius metidjensis.

Snail species

P. metidjensis

Snail Origin

Spain

Spain

S. bovis origin

Spain

Spain

Number of miracidia per snail

2 3 4 5 5 5 5 8 9 10 10 10 10 10 15 50 5, 10 or 20 5, 10 or 20 5

5 10

Number of surviving or exposed' snails

87 46

21 50 39 40 43 19 43 18 7 15 24 92 4 3 258' 109' 25' 25' 25'

% snails infected with S. bovis from

NMZ' 17.2 17.6 14.3 44 28.2 17.5 11.5 52.5 72 28 100 73 83.3 75 25 100 69.4 44.2 20

64 24

SMZ3

References

SSZ4 Ramajo-Martin (1972)

Ramajo-Martin (1978)

I

r

0

Sampaio-Silva et al. (1975)

z rn

rn ' I b

!-

5,

Portugal

Morocco

Spain

Portugal Portugal Morocco

Sardinia (Italy) Morocco Iran Sudan

Spain Spain

Sudan Kenya

Portugal

'

Spain

Number of exposed snails. *North Mediterranean zone. South Mediterranean zone. South Saharan zone.

5, 5, 5, 5,

10 20 20 10 or 20 5 5 10 10 20 20 10 or 20 10 or 20 10 or 20 10 or 20 5 10 5

5 5 5 10 5, 10 or 20 5, 10 or 20

25' 25' 25' ?

25' 25' 25' 25' 25' 25' ? ? ? ?

94 71 ? ? ?

170 71 ? ?

72 8 32 73.3 68 12 52 24 80 20 62.9 78.6 29.2 14.3 45.7 86 0

Southgate et al. (1984) Sampaio-Silva et al. (1975)

m c i 0

z

Ramajo-Martin (1978) Southgate et al. (1984) Touassem and Jourdane (1986) Southgate and Knowles (1975a)

0 0 5.9 35.2 0 0

Touassem and Jourdane (1986) Southgate et al. (1984)

5 s cn

A

Table 3 Snail infection experiments in the genus Bulinus of the truncatusltropicus group.

0 Q)

Snail species

B. natalensis

B. octoploidus

Snail origin

South Africa Ethiopia

S. bovis origin

Ethiopia Ethiopia

B. permembranaceus

Kenya

Morocco

B. trigonus

Ethiopia Kenya Mauritania Uganda Uganda Kenya Uganda Kenya Sudan Tanzania

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Morocco Iran Iran Kenya Kenya Tanzania

Uganda

Kenya


5 15-20


0 6.3

16 48

5-6

?

3

? ? ?

23 25 25 28 20-25' 20-25' 20-25' 20-25' 25

Loand Lemma (1975)

0 8.3

15

41 47 24

References

NMZ2 SMZ3 SSZ4

44

10-15 20

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

% snails infected with S . bovis from

Frandsen (1979)

0

Southgate and Knowles (1975a)

21.9 12.7 95.7 0 0 0

0

30.4 20 32 14.3 13 38 17 50 12

Southgate et al. (1980) Mutani et al. (1983)


I rn

' I

b !-

B. tropicus

South Africa South Africa South Africa Zimbabwe

Sardinia (Italy) Morocco Iran Kenya

Tanzania

Tanzania

Zambia

Tanzania

Ethiopia Kenya

Tanzania Tanzania

South Africa

Tanzania

5 5 5 5 5 5 5 4 10 30 5 5 5 5 5 5

5 B. truncatus Mediterranean zone

Portugal

Spain 5, 5,

Egypt

Spain

Libya

Spain

5, 5,

5 10 20 10 or 20 10 or 20 5 10 20 10 or 20 10 or 20

? ? ?

0

251 20-251 20-251 20-251

9

0 0 0 0 0 0 0 0 0 0 0 0 0

9

30 24 20-251 20-251 20-251 20-251 20-25' 20-251 20-251 25' 251 25' 168' 90' 251 25' 251 1501 331

0 0 0 0

52.1 100 85 67.3 74.4 55.5 70 94.4 55.1 50

W

Southgate et al.

c

Mutani et al. (1983) (1985a)

0 Z

=!

4 Kassuku et al. (1986) Kinoti (1964a) Mutani et al. (1983)

$ 2 0 $

5

Simon-Vicente et al. (1975) Ramajo-Martin (1978) Simon-Vicente et al. (1975) Ramajo-Martin (1978) (continued)

~

0 4

a

Table 3 Continued.

UI

Snail species

Snail origin

S. bovis origin

Tunisia

Spain

Israel Egypt Iraq Libya Morocco Sardinia (Italy) Iran Israel Egypt Iraq Libya

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Morocco Morocco Morocco Morocco Morocco

Morocco

Morocco

Israel

Israel

Iran Portugal

Iran Iran

Number of miracidia per snail 5 10 5 5 5 5 5 5 5 5 5 5 5 6 5 5 5, 10, 15 or 20 5, 10, 15 or 20 60 2-4 2-4

Number of surviving or exposed' snails 57 53 ?

20 18 23 43 99 ? ?

14 45 101 24 54

YOsnails infected with S. bovis from

NMZ2 SMZ3 SSZ4 Touassem and Jourdane (1986) Southgate and Knowles (1975a)

61.4 56.6 0 15 5.6 60.9 30.2 21.2

0 0 7.1 4.5 32.7 91.6 31.5

?

90 36

?

58.6

?

87 53.3 60

30

30 20

References

Frandsen (1979) Southgate and Knowles (1975a) Frandsen (1979) Lengy (1962)

r 0 Arfaa ef al. (1967)

z rn rn -4 L

r

630ch3.3d Southgate and Knowles (1975a)

B. truncatus South Saharan zone

Iraq

Iran

5

59

50.8

Israel Libya Morocco Sardinia (Italy) Egypt Egypt

Iran Iran Iran Iran Iran Ethiopia

5 5 5 5 5 20

43 234 28 64 40

15

Israel Libya Morocco Sardinia (Italy) Tunisia

Kenya Kenya Kenya Kenya Sudan

5 5 5 5 5 10

23 17 25 20 160 53

13 47.1 16 45 15 41.5

Chad

Spain

5 10 20 5, 10 or 20

25' 25' 25' 130'

Sudan Ethiopia Kenya Mauritania Malawi Senegal

Morocco Morocco Morocco Morocco Morocco Morocco

86 22.2 25 11 0

?

?

30 50 106 91 40 57

25 33.5

Lo and Lemma (1975) Southgate and Knowles (1975a) Touassem and Jourdane (1986) Simon-Vicente et al. (1975)

100

64.5

0 23.3 18 0.9 86.4 87.5 93

Ramajo-Martin (1978) Southgate and Knowles (1975a) Frandsen (1979)

(continued

Table 3 Continued.

Snail species

B. coulboisi (syn. B. truncatus)

Snail origin

S. bovis origin

Zimbabwe Uganda

Morocco Morocco

Uganda Uganda Uganda Tanzania Kenya

Sardinia (Italy) Morocco Iran Tanzania Tanzania

Ethiopia

Tanzania

Sudan Zambia Gabon

Tanzania Tanzania Senegal

Senegal

Senegal

Uganda

Kenya



5 5 or 6

28

5 5 5 4-5

25 24 19

5 5 5 5 5 5 5


NMZ'

40

SMZ3 SSZ4

3.6 0

?

20-25l 20-25l 20-25l 20-25l 20-25l 20-25l 50

References


24 4 10.5 0 50

Kinoti (1 964a) Mutani et al. (1983)

60 0 19 0 0 48

3-4

19

63.6

5

23

8.7

Southgate et al. (1985b) Diaw and Vassiliades (1987) Southgate and Knowles (1975a)

?

5 Z

rn

B. guernei (syn. B. truncatus)

Senegal

Senegal

5

89

64

Southgate et al. (1985b)

rn

-i

b !-

B. sericinus (syn. B. truncatus)

The Gambia

Ethiopia

5

45

Ghana Nigeria Cameroon Ghana Nigeria Chad Cameroon Ghana Cameroon Ghana

Sardinia (Italy) Sardinia (Italy) Morocco Morocco Morocco Iran Iran Iran Kenya Tanzania

5 5 5 5

59 14 27 98 38

5

41 95 39 15

Saudi Arabia Saudi Arabia Saudi Arabia Saudi Arabia


5 5 5 5

17 18 25 21

5 5 5 5 5

2.2 57.6 100

Loand Lemma (1975) Southgate and Knowles (1975a)

7.4 59.2 42.1 100 12.2 46.4

5

33.3 53.3 82.3

Southgate et al. (1980) Southgate and Knowles (1975a)

11.1 20

23.8

Number of exposed snails. North Mediterranean zone. South Mediterranean zone. South Saharan zone.

A A A

Table 4 Snail infection experiments in the genus Bulinus of the reticulatus group.

Snail species

B. reticulatus

B. wrighti

Snail origin

Kenya Kenya Kenya Kenya


Saudi Arabia Saudi Arabia Saudi Arabia Saudi Arabia Yemen Oman Yemen

Sardinia (Italy) Morocco Iran Kenya Kenya Tanzania Tanzania

Oman

'

S. bovis origin

Number of exposed snails. 'North Mediterranean zone. South Mediterranean zone. South Saharan zone.

Senegal


Number of surviving or exposed' snails 8 12 8 7

5 5 5 5

5 5 5 5 5 5

30 86 8 25 16' 14 11 20-25' 20-25' 19


Nh4Z'

SMZ3

References

SSZ4

50


8.3 25 100

80 34.9 62.5 56

12.5 100 36.4 92 84 47.4

Southgate et al. (1985a) Southgate et al. (1980) Mutani el al. (1983) Southgate et al. (1985b) ?

z

0 z rn

m

-i

b

r

Table 5 Snail infection experiments in the genus Bulinus of the forskalii group. Snail species

Snail origin

S. bovis origin



% snails infected with

NMZ'

SMZ3

SSZ4

Madagascar Madagascar Madagascar

Sardinia (Italy) Morocco Iran

5 5 5

3 2 40

33.3

Cameroon Cameroon Cameroon Cameroon


5 5 5 5

25 22 23 3

80

B. canescens

Zambia

Tanzania

5

20-25'

0

B. cernicus

Mauritius

Kenya

15

43

53.5

B. crystallinus

Angola Angola Angola Angola Angola

Sardinia (Italy) Morocco Iran Kenya Tanzania

5 5 5 5 5

24 79 39 13 48

Cameroon Nigeria Senegal

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy)

5 5 5

? ?

B. bavayi

B. camerunensis

B. forskalii

25

References

S. bovis from


50 47 100 69.9 100

11.9

Rollinson and Wright (1984) Southgate and Knowles (1975a)

1.3 17.9

30.8 31.3 0 0 12

Mutani et al. (1983)

Southgate et al. (1980) Southgate and Knowles (1975a)

continued

Table 5 Continued.

Snail species

B. scalaris

Snail origin

S. bovis origin

Kenya Nigeria Senegal QYPt Cameroon Nigeria Senegal Cameroon Ethiopia Senegal Zaire

Morocco Morocco Morocco Morocco Iran Iran Iran Kenya Kenya Kenya Tanzania

Tanzania

Tanzania

Tanzania

Tanzania

Gabon Senegal

Senegal Senegal

Zimbabwe Zimbabwe Zimbabwe


' Number of exposed snails. 'North Mediterranean zone. South Mediterranean zone. South Saharan zone.


5 5 5 5 or 6 5 5 5 5 5 5 5 5 5



NMZ'

SMZ3

?

45 25 17 12 25 20-25' 20-25' 19 17 21 24 9 96

5 5 5

? ? ?

0

0 0

P

Frandsen (1979) Southgate and Knowles (1 975a) 30.8 100 68 100 85 52.9 63.2 64.7 100 41.7 66.7 48.9

?

4- 5 5 5 5 5 3-4

a a

SSZ4

0 0 37.5 0 0 6.7 8

? 24 ?

References

Mutani et al. (1983) Southgate et al. (1980) Kinoti (1964a) Mwambungu (1988) Southgate et al. (1985b) Diaw and Vassiliades (1987) Southgate and Knowles (1975a)

I

5

P v)

i W

c

-I

0

z

Table 6 Snail infection experiments in the genus Bulinus in the africanus group.

Snail species

B. abyssinicus B. africanus

Snail origin

S. bovis origin

Ethiopia Somalia

Ethiopia Tanzania

South Africa Ethiopia South Africa Ethiopia South Africa Ethiopia Ethiopia Kenya

Sardinia (Italy) Sardinia (Italy) Morocco Morocco Iran Iran Ethiopia Tanzania

Tanzania

Tanzania

Number of miracidia per snail 5

5

5 5 5 5 5 5 20 5 5 5 5 5 5 4 4-6



? ? ? ? ? 20 4 20-25' 20-25' 20-25' 20-25' 20-25' ? 35

References

0

a

0

8

62.5 0

Lo and Lemma (1975) Mutani et al. (1983) Southgate and Knowles (1975a)

0 100 67 83 83 100 100 83.3 65.7

Lo and Lemma (1975) Southgate et al. (1980) Mutani et al. (1983)

0 0

cn

-i

N M Z ~ SMZ~ SSZ~

8 20-25' ?

%

0 0 0 0

Kassuku et al. (1986) Kinoti (1964a) continued

51D

0

s

Table 6 Continued

Snail species

B. globosus

Snail origin

S . bovis origin

Zimbabwe



Sardinia (Italy)

Nigeria South Africa Uganda Zaire Zimbabwe Zimbabwe albino Uganda South Africa Zambia Zimbabwe

Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Sardinia (Italy) Morocco Morocco Morocco Morocco Morocco Iran

Nigeria South Africa Uganda Zaire Zimbabwe Zimbabwe Zimbabwe albino

Iran Iran Iran Iran Ethiopia Kenya Kenya

6 8-9 10-12 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5 5


Nh4Z2

SMZ3

20 20 23 ?

47 ? ? ? ? 37


0 29.8 0 0 0 0

27 ? ? ? ?

0 0 0 0

13 24 9

SSZ4 95 100 100

10.8 80 0 0 0 0 68.2

5 ? ? ? ?

References

Frandsen (1979) Southgate and Knowles (1975a)

I

Lo and Lemma (1975) 8.3 Southgate and Knowles 11.1 (1975a) 0

I 0 z rn

3 b

r

South Africa Ghana Uganda Tanzania

Kenya Kenya Kenya Tanzania

5 5 5 10-12 5 5

Zimbabwe South Africa Kenya Kenya albino

Tanzania Tanzania Tanzania Tanzania

B. jousseaumei

Senegal

Senegal

B. nasutus

Tanzania

Tanzania

5 5 5 5 5 5 5 5 5 5 5 5 5 4-5 10-12 15 100 5

5

13 17 23 24 ?

20-251 20-25' 20-25' 22 23 20 25 14 25 20-251 20-251 20-25' 20-251 ?

6 29 28 17

30.8 11.8 33 4.1 Kinoti (1964a) 20 Southgate and Knowles (1975a) Mutani et al. (1983) 0 5 4

63.6 0 0 20 100 32 71 28 40 67 0

?

0 0 0 0 0

20-251

0

Mwambungu (1988) Southgate et al. (1980) Mutani et al. (1983)

Southgate et al. (1985b) Kinoti (1964a)

Southgate and Knowles (1975a) Mutani et al. (1983) continued

--.

Table 6 Continued.

Snail species

B. obtusispira

B. ugundae

Snail origin


OD

Number of % snails infected with surviving S. bovzk from or exposed’ snails NMz’ S M Z ~ S S Z ~

Madagascar Madagascar Madagascar


5 5 5

? ? ?

0

Uganda

5 5 5

? ? 25

0

Uganda

Sardinia (Italy) Morocco Kenya

Senegal

Senegal

5

22 15

Uganda B. umbilicatus

S. bovis origin

2

3-4

References


0 0


0 100

4.5 Southgate et ul. (1985b) 13.3 Diaw and Vassiliades (1987)

Number of exposed snails.

’North Mediterranean zone. South Mediterranean zone. South Saharan zone.

6z

rn

m

-i

b

r

119


Table 7 The species of Planorbarius and Bulinus, and their relationships as natural and experimental hosts for Schistosoma bovis of the North Mediterranean zone (NMZ), of the South Mediterranean zone (SMZ) and of the South Saharan zone (SSZ). The mollusc species are named alphabetically within each group. Natural host Mollusc Genus Planorbarius P. corneus P. metidjensis Genus Bulinus (truncatus/ tropicus group) B. angolensis B. depressus B. hexaploidus B. Iiratus B. natalensis B. nyassanus B. octoploidus B. permembranaceus B. succinoides B. transversalis B. trigonus B. tropicus B. truncatus B. yemenensis Genus Bulinus (reticulatus group) B. reticulatus B. wrighti Genus Bulinus uorskalii group) B. barthi B. bavayi B. beccarri B. browni B. camerunensis B. canescens B. cernicus B. crystallinus B. forskalii B. scalaris B. senegalensis Genus Bulinus (africanus group) B. abyssinicus

Experimental host

NMZ

SMZ

SSZ

NMZ

SMZ

SSZ

NF Yes

NL No

NL NL

NT Yes

NT No

NT Yes

NL NL NL NL NL NL NL NL NL NL NL NL Yes NL

NL NL NL NL NL NL NL NL NL NL NL NL Yes NL

NF NF NF NF NF NF Yes NF NF NF NF Yes Yes NF

NT NT NT NT NT NT NT NT NT NT Yes No Yes NT

NT NT NT NT NT NT NT No NT NT No No Yes NT

NT NT NT NT Yes NT Yes NT NT NT Yes No Yes NT

NL NL

NL NL

NF NF

Yes Yes

Yes Yes

Yes Yes

NL NL NL NL NL NL NL NL NL NL NL

NL NL NL NL NL NL NL NL NF NL NL

NF NF NF Yes NF NF NF NF Yes NF Yes

NT Yes NT NT Yes NT NT Yes Yes No NT

NT Yes NT NT Yes NT NT Yes Yes No NT

NT NT NT NT Yes No Yes Yes Yes NT NT

NL

NL

Yes

NT

NT

Yes

(continued)

120

H. MONE ETAL.

Table 7 Continued.

Natural host Mollusc B. africanus B. globosus B. hightoni B. jousseaumei B. nasutus B. obtusispira B. obtusus B. ugandae B. umbilicatus

Experimental host

NMZ

SMZ

SSZ

NMZ

SMZ

SSZ

NL NL NL NL NL NL NL NL NL

NL NL NL NL NL NL NL NL NL

Yes Yes NF NF NF NF NF Yes NF

No Yes NT NT NT No NT No NT

No Yes NT NT

Yes Yes NT No No NT NT Yes Yes

NT No NT No NT

NF = the species of mollusc has not been found infected yet in that zone; NL = the species of mollusc do not live in that zone; NT =the host-parasite association has not been tested.

Figure I Geographical distribution of Planorbarius metidjensis (shaded area). (Data obtained from Brown, 1994.) 0 = Countries in which Planorbarius metidjensis has been found naturally infected by S. bovis.

Figure 2 Geographical distribution of Bulinus belonging to the truncatus (shaded area)/tropicus (hatched area) group. (Redrawn with permission from Brown, 1994, Figures 126 and 127.) 0 = Countries in which the molluscs of the truncatusltropicus group have been found naturally infected by S. bovis.

Figure 3 Geographical distribution of the molluscs of the Bufinus reticulatus group (shaded area). (Data obtained from Brown, 1994.)

Figure 4 Geographical distribution of the molluscs of the Bulinus forskalii group (shaded area). (Redrawn with permission from Brown, 1994, Figure 128.) O=Countries in which the molluscs of the forskalii group have been found naturally infected by S.bovis.

Figure 5 Geographical distribution of the molluscs of the Bulinus africanus group (shaded area). (Redrawn with permission from Brown, 1994, Figures 124 and 125.) 0 = Countries in which the molluscs of the africunus group have been found naturally infected by S. bovis.

DISTRIBUTION OF SCH/STOSOMA BOWS

123

test or the unpaired t-test were applied on the arcsine-transformed data. For each prevalence, one set of arcsine-transformed data was calculated.

3. THE NATURAL MOLLUSC INTERMEDIATE HOST SPECTRUM

The natural intermediate host spectrum of Schistosoma bovis is wide: S. bovis may naturally infect molluscs belonging to two genera, Planorbarius and Bulinus. Within these genera, one species of Planorbarius (P. metidjensis) and ten species of Bulinus may act as intermediate hosts, showing a wide host spectrum tolerance of S. bovis for mollusc intermediate hosts. The species belonging to the genus Bulinus are usually divided into four groups: truncatusltropicus, reticulatus, forskalii and africanus. In each group, the recent classification of Brown (1994) was used. S . bovis may infect Bulinus species belonging to three of the four groups (Table 7). The comparative intermediate host spectra of S. bovis between the three geographical zones showed that: the North Mediterranean populations of S. bovis use two species of mollusc, P. metidjensis and B. truncatus; the South Mediterranean populations of S . bovis use only B. truncatus; and the South Saharan populations use ten species of Bulinus. B. truncatus is the only species acting as an intermediate host in the three zones.

4. GEOGRAPHICAL DISTRIBUTIONS OF THE MOLLUSC

INTERMEDIATE HOSTS

The geographical distribution of P. metidjensis is presented in Figure 1. It is limited to the two Mediterranean zones, the North Mediterranean zone to the Iberian peninsula, and the South Mediterranean zone to Morocco and Algeria. The geographical distributions of the Bulinus species are presented in Figures 2-5. The truncatusltropicus group occurs in the three geographical zones, and it has a Mediterranean (North and South) and Pan-African distribution (Figure 2). The reticulatus group has a sparce geographical distribution in the South Mediterranean zone (Arabia peninsula) and in the South Saharan zone (Figure 3). The forskalii group has a sparce geographical distribution in the South Mediterranean zone (Egypt) but is widely present in the South Saharan zone, where it has an Afro-intertropical geographical distribution extending to Yemen and some islands of the Indian Ocean (Figure 4). The africanus group has a strictly South Saharan Afro-intertropical distribution including Madagascar (Figure 5).

124

H. MONE ETAL.

5. GEOGRAPHICAL DISTRIBUTION OF S. BOWS

The natural geographical distribution of S . bovis is presented in Figures 1-5, according to the mollusc group, and in Table 7. It runs from the latitude 43 ON crossing the North of Corsica, France, to the latitude 13 "S crossing the very south of Zaire, and from the longitude 17.7"W crossing the Green Cap in Senegal to the longitude 63.2"E crossing the very east of Iran. It has been found in countries in all three zones (3 countries in the North Mediterranean zone, 4 countries in the South Mediterranean zone and 12 countries in the South Saharan zone). The number of regions where S . bovis is present is smaller than the number of regions where the mollusc intermediate hosts are found. In some other regions, we may just suppose an actual transmission of S . bovis because its presence in cattle does not imply that the definitive host has been infected locally (Figure 6 ) .

Figure 6 Countries where S. bovis is probably present (0).In these countries, S . bovis was found in vertebrate definitive hosts whose geographical origin was uncertain.


125

6. THE EXPERIMENTAL MOLLUSC INTERMEDIATE HOST SPECTRUM

The experimental intermediate host spectrum of S. bovis is wider than the natural one: among the 24 species of mollusc tested, S . bovis may experimentally infect one species of Planorbarius ( P . metidjensis) and 16 species of Bulinus belonging to the four groups (Table 7). The comparative intermediate host spectra of S . bovis between the three geographical zones showed that the North Mediterranean populations of S. bovis may use ten species of molluscs belonging to two different genera and to the four groups of Bulinus, the South Mediterranean populations of S. bovis may use eight species of molluscs all belonging to the four groups of the genus Bulinus, and the South Saharan populations may use 16 species of molluscs belonging to two different genera and to the four groups of Bulinus. B . truncatus for the truncatusltropicus group, B. reticulatus and B. wrighti for the reticulatus group, B . camerounensis, B. crystallinus and B. forskalii for the forskalii group, and B. globosus for the africanus group have been experimentally infected with S . bovis populations from the three zones.

7. COMPATIBILITY IN THE MOLLUSC-S. BOWS ASSOCIATION

A total of 294 prevalences were available, including 188 population hostpopulation parasite systems. Experimental prevalences 44, 124, 14, 39 and 73 obtained for P . metidjensis, and the truncatusltropicus, reticulatus, forskalii and africanus groups, respectively, are presented in Tables 2-6 for the three geographxal zones. For each species of mollusc, the order in which these prevalences are presented was always the same: firstly, the prevalences concerning the S. bovis populations of the North Mediterranean zone; then the prevalences concerning the S. bovis populations of the South Mediterranean zone; and, finally, the prevalences concerning the S. bovis populations of the South Saharan zone. Transformed prevalences (p’) 9, 88, 11, 34 and 46 obtained for each population host-population parasite system for P . metidjensis, and the truncatusltropicus, reticulatus, forskalii and africanus groups, respectively, were used for the analysis of the host-parasite compatibilities. The mean transformed prevalences are presented in Table 8 together with the ANOVA comparisons. When considering the influence of the mollusc group on compatibility, the results showed that the compatibilities were significantly different among the

Table 8 Transformed prevalences of Schistosoma bovis from the North Mediterranean zone (NMZ), the South Mediterranean zone (SMZ) and the South Saharan zone (SSZ) according to the mollusc group.

Mollusc group

Genus Planorbarius P. metidjensis Genus Bulimus truncatusltropicus group reticulatus group forskalii group ajiricanus group ANOVA

NMZ

ssz

SMZ

N

Mean f SE

N

Mean fSE

N

Mean fSE

4

0.57 f0.19

2

0.00 f 0.00

3

0.16 f0.16

20 0.62 f 0.09 2 0.95 f 0.16 7 0.35 f 0.16 9 0.04 f0.04 F= 5.746; P = 0.002 S

39 4 15

0.45 f 0.07 0.59 f 0.13 0.39 f 0.12 17 0.12 f0.07 F = 2.72; P = 0.036 S

27 0.37 f0.06 5 1.02 f0.20 12 0.89f0.13 20 0.58f0.12 F= 5.93; P = 0.0004 S

ANOVA

F = 2.75; P = 0.14 F = 2.45; P = 0.09 S F= 1.48; P=0.28 F=5.00; P=O.O13 S F=8.79; P=0.0006 S

S = significance. I

DISTRIBUTION OF SCHISTOSOMA BOVIS

127

groups whatever the S. bovis geographical zone: 1. for the North Mediterranean populations of S. bovis, the reticulatus, and truncatusltropicus groups and P . metidjensis showed the highest compatibilities; 2. for the South Mediterranean populations of S. bovis, the compatibilities were the highest for the reticulatus, truncatusltropicus and forskalii groups and null for P . metidjensis; 3 . for the South Saharan populations of S. bovis, the reticulatus, forskalii and africanus groups showed the highest compatibilities. When considering the influence of the geographical origin of S. bovis on compatibility, the results showed that the compatibilities were significantly different only in the forskalii and the africanus groups as shown below. 1. Despite the few data available, P . metidjensis was highly compatible with

2. 3. 4.

5.

the North Mediterranean population of S. bovis but refractory with the South Mediterranean populations, and slightly compatible with the South Saharan populations (see Table 8). In the truncatusltropicus group, the highest prevalence was found with the North Mediterranean populations of S. bovis. The reticulatus group was highly compatible with the three geographical populations of S. bovis. The forskalii group was compatible with the three geographical populations, with a significant highest prevalence in the South Saharan populations of S. bovis. The significant difference came from the comparison between the South Saharan populations of S. bovis and the South Mediterranean populations (post hoc Scheffe comparison procedure: P = 0.03). Notice that the prevalences between the two Mediterranean zones, with only the Sardinian population as member of the North Mediterranean zone, were not significantly different (see Tables 5 and 8). The africanus group was compatible with the South Saharan geographical populations of S. bovis, but only slightly compatible with the populations from the other two zones. The difference was highly significant for the comparison between the South Saharan populations of S. bovis and the North Mediterranean populations (post hoc Scheffe comparison procedure: P = 0.006), and for the comparison between the South Saharan populations of S. bovis and the South Mediterranean populations (post hoc Scheffe comparison procedure: P = 0.004). Notice that the prevalences of the two Mediterranean zones, with only the Sardinian population as member of the North Mediterranean zone, were very low and not significantly different (see Tables 6 and 8).

When considering the North Mediterranean zone, the populations of S. bovis, peculiarly, were very heterogeneous in their compatibilities towards

128

H. MONE ETAL.

P . metidjensis, and may be separated into the highly compatible Spanish populations (0.76 f 0.005) and the refractory Sardinian populations (see Table 2). Similarly, for the truncatus-tropicus group, the Spanish populations (0.91 f 0.05; N = 5) had a higher compatibility than the Sardinian populations (0.52 f 0.13; N = 15) but the difference was just below being significant ( t = 2.08; P = 0.05) (see Table 3). For the other three groups, reticulatus, forskalii and africanus, only Sardinian populations of S. bovis were tested for the North Mediterranean zone (see Tables 4-6) and no comparison with the Spanish populations could be made with our data set.

8. THREE MAIN POPULATIONS OF S. BOWS

The natural and experimental intermediate host spectrum and host-parasite compatibility results between S. bovis and its intermediate hosts showed that our hypothesis, according to which the two natural barriers (the Mediterranean Sea and the Sahara) played a role on the genetic flow between the different populations of S. bovis, has to be rejected for the Mediterranean barrier but that it can be accepted for the Saharan barrier. The intrazone variability was clearly high for the North Mediterranean populations confronted with P. metidjensis, and suggests a separation between the highly compatible Spanish populations and the refractory Sardinian populations, which behaved like South Mediterranean populations. The interzone variabilities between the North Mediterranean zone and the South Mediterranean zone were similar enough to reject the hypothesis concerning the role played by the Mediterranean barrier. The interzone variabilities between the South Mediterranean zone and the South Saharan zone were high enough to accept the separation according to the Saharan barrier. Thus, three main geographical populations of S . bovis exist but they are different from those proposed by our hypothesis: from the North to the South, we find the Iberian populations (Spanish and Portuguese), the Mediterranean populations (including the Sardinian populations), and, finally, the South Saharan populations. The Iberian populations of S. bovis use exclusively P . metidjensis in the natural conditions; their host spectrum is thus restricted to one species. At the same time, the reanalysed compatibilities according to these three new geographical zones (Figure 7) showed that the Iberian populations are as compatible with B. truncatus as with P . metidjensis, and both compatibility values are high. This result agrees with those of Southgate and Knowles (1975~)whose results could not be included in our data set because the origins of the snails were not precise. However, these authors were the sole authors who showed experimentally that the Spanish population of S. bovis was not


129

1.m 1.20 1.00

0.80 0.60 0.40

0.20 0.00

Figure 7 Comparative compatibilities of S. bovis from the Iberian zone (a),the Mediterranean zone (0)and the South Saharan zone @) for the different groups of molluscs.

compatible with the africanus group, that it had a very poor compatibility with the forskalii group and that it had a reasonable good compatibility with the reticulatus group. For our results, the deletion of the Sardinian populations from our previous North Mediterranean zone made the prevalences higher for the Iberian populations (compare Table 8 and Figure 7). The Mediterranean populations, including the Sardinian populations of S. bovis, use exclusively B. truncatus in natural conditions; their natural host spectrum is also restricted to one species. In experimental conditions, they are refractory to P . metidjensis (Figure 7 ) ; however, they are compatible with all other groups of Bulinus: the compatibility is the highest with the truncatusltropicus group and weaker with the species belonging to the africanus group which proves to be restricted to the S. bovis populations of the South Saharan zone. The inclusion of the Sardinian populations in the new redefined Mediterranean zone did not alter the pattern (compare Table 8 and Figure 7). The South Saharan populations of S. bovis benefit from a wide natural host spectrum: the natural hosts belong to three groups of Bulinus the truncatus/tropicus, forskalii and africanus groups. In experimental

130

H. MONE ETAL.

conditions, S. bovis successfully infected some species from all groups of molluscs (Figure 7), but preferentially the reticulatus, the forskalii and the africanus groups. The compatibility is the lowest with the genus Planorbarius, which appears to be restricted to S. bovis from the Iberian zone. This situation is very different from that occurring in the Mediterranean zone where the truncatusltropicus group plays the lead role in the transmission of S. bovis. The South Saharan populations of S. bovis are slightly compatible with P.metidjensis.

9. PALEOBIOGEOGRAPHICALSCENARIO OF S. BOVlS

From these results, one possible scenario of the natural paleobiogeography of the S. bovis species complex could be proposed (Figure S), despite the lack of any fossils of schistosomes and the wide vertebrate definitive host spectrum (see Mouahid, 1984), creating possible lateral transfers between hosts favoured by animal migrations (Combes, 1995). The time scale of the history of S. bovis could not be determined, but the knowledge on both intermediate host spectrum and compatibility allows us to propose a three-step but probably continuous history of this species. The first step is the South Saharan origin of S. bovis, which is not in doubt, even though the exact local origin is unknown (Combes, 1990; Combes et al., 1991; Despres et al., 1992). It will be assumed that the origin was in the West of Africa. As early as its origin, S . bovis was confronted with a wide spectrum of molluscs belonging to the genus Bulinus and consequently coevolved with them. The extension of the geographical distribution to the South, East and West did not alter the intermediate host spectrum. The very low compatibilities between the South Saharan populations of S. bovis and Planorbarius may be explained by the absence of this mollusc in the South Saharan region. The second step, occurring in a more recent period, is the extension of S. bovis to the Mediterranean zone. The North migration was accompanied by a drastic change in the host spectrum, which was reduced to one species of mollusc. As this species, B. truncatus, also existed in the South Saharan zone, S. bovis did not necessarily have to adapt to it, as suggested by the similar compatibilities existing between the Mediterranean and the South Saharan populations of S. bovis with B. truncatus. The low compatibilities between the Mediterranean populations of S. bovis and the mollusc species of the africanus and forskalii groups corroborate the existence of an adaptation of the Mediterranean populations to a host spectrum restricted to one species. In this second step, the North-West and the North-NorthEast migrations have to be analysed separately.

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Figure 8 The three steps of the paleobiogeographical scenario of S. bovis.

The North-West extension of S. bovis stopped in Morocco owing to the natural barrier of the Gibraltar Strait. Indeed, the few data available on the time-scale history of S. bovis are compatible with a recent origin of S. bovis in this region. According to recent schistosome molecular phylogenies, S. bovis emerged as a new species between 1 and 4 and between 1 and 7 million years ago, respectively, based on nuclear and mitochondria1 DNA (Despres et al., 1992). Recently, a study on the relative phyletic relationships in schistosomes using RAPD markers has even suggested a very recent origin of S. bovis, that is, the possibility that this species has been transferred from man to livestock (Barral, 1996). These results from molecular biology thus suggest it is unlikely that S. bovis originated in Africa before or even during the Messinian period (also called the ‘Salt crisis’), which lasted from 6 to 5.5 million years ago, and where the Mediterranean Sea level dropped more than 1000 metres, thus blocking the connection between the Mediterranean

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Sea and the Atlantic Ocean (Van der Zwaan, 1982) at the Strait of Gibraltar. Furthermore, during this period, the Iberian peninsula-North West African interchange of land vertebrates was followed by massive extinctions of these vertebrates, removing the possibility for them to act as important hosts for any parasite evolution (Jaeger et al., 1987; Jaeger and Hartenberger, 1989). It is important to note there that the Moroccan populations of S . bovis have been always detected in B. truncatus despite the presence of Planorbarius. The North-North-East extension of S . bovis used the Middle Eastern passage to invade the Southern part of Europe and Sardinia. This extension could have been favoured by human migration with cattle to Europe and to the Mediterranean islands. For instance, humans are known to have introduced domestic species in some Mediterranean islands as early as 7000 BP (Blonde1 and Vigne, 1993). The third, last and more recent step was the colonization of the Iberian peninsula by S. bovis. The intermediate host spectrum of the schistosome ‘reopened’ and S. bovis captured a new host belonging to a different genus, the genus Planorbarius. This capture of a new genus would have been harder than the capture of any other new species in the same genus owing to its remote phylogenetic position from the molluscs of the genus Bulinus (Hubendick, 1955). The strict non-compatibility between the Mediterranean populations of S. bovis and P. metidjensis, and the slight compatibility between the South Saharan populations of S. bovis and P. metidjensis, compared to the high level of compatibility between the Iberian populations of S. bovis and P. metidjensis, suggest the existence of a local adaptation of the Iberian S. bovis strain to Planorbarius, which isolated the Iberian populations of S. bovis from the remainder of the Mediterranean and Afrotropical populations. This local adaptation has been proposed by Mouahid and Thtron (1987) comparing the cercarial production of a Spanish population of S . bovis among B. truncatus, B. wrighti and P. metidjensis. These authors showed that, with a lower productivity compensated by a lengthening of the production period, S . bovis was better adapted to P. metidjensis than to the species belonging to the genus Bulinus because the parasite exploited this mollusc host optimally.

10. CONCLUSION

From our results, the three major groups of S. bovis populations that could be determined were the Iberian, the Mediterranean and the South Saharan populations. The Saharan barrier may have played a role in the gene flow between the different populations of S. bovis but not the Mediterranean one.

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More work should be done with the different populations of S . bovis, especially with those existing around the Mediterranean Sea, because not all of the interzone combinations have been tested. Despite the data originating from different authors working under different conditions, we believe that the host-parasite compatibility may constitute a tool for researchers to extend knowledge on the evolutionary biogeography of hosts and parasites.

ACKNOWLEDGEMENTS We gratefully acknowledge Drs V. R. Southgate (The Natural History Museum, London, UK), S. Mas-Coma (University of Valencia, Spain) and C. Combes (University of Perpignan, France) for their comments and constructive criticism. This work received financial support from the UNDP World Bank WHO Special Program for Research and Training in Tropical Diseases and the CNRS (Sciences de la Vie).

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The Larvae of Monogenea (Platyhelminthes) Ian D. Whittington’a2. Leslie A . Chisholm’ and Klaus Rohde3

’Department of Parasitology. The University of Queensland. Brisbane. Queensland 4072 Australia; Heron Island Research Station of The University of Queensland. Great Barrier ReeA via Gladstone. Queensland 4680. Australia; School of Biological Sciences. Division of Zoology. University of New England. Armidale. New South Wales 2351. Australia

Abstract ................................................................... 1. Introduction .............................................................. 2. General Morphology ..................................................... 2.1. Monopisthocotylea ................................................. 2.2. Polyopisthocotylea .................................................. 3. Haptoral Sclerites ........................................................ 3.1. Summary .......................................................... 4. Ciliated Cells ............................................................. 4.1. Monopisthocotylea ................................................. 4.2. Polyopisthocotylea .................................................. 4.3. Summary .......................................................... 5. Epidermis ................................................................ 5.1. Monopisthocotylea ................................................. 5.2. Polyopisthocotylea .................................................. 5.3. Summary .......................................................... 6. Terminal Globule ......................................................... 6.1. Monopisthocotylea ................................................. 6.2. Polyopisthocotylea .................................................. 6.3. Possible Functions .................................................. 6.4. Summary .......................................................... 7. Glands................................................................... 7.1. Monopisthocotylea ................................................. 7.2. Polyopisthocotylea .................................................. 7.3. Function ........................................................... 7.4. Summary .......................................................... ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

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Copyright 0 2000 Academic Press A / / rights of reproducrion in any form reserved

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8. Protonephridia . . . . . . . . , . , . , . . . . . . . , . . . . . . . . . . . . . , . . . . . . . . . . . . . . . 177 8.1. Monopisthocotylea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.78 8.2. Polyopisthocotylea.. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 180 8.3. Summary .......................................................... 182 . . . . . .. 183 9. Sense Organs.. . . . . . . . . . .. .. . . . . , , , . . .. . .. .. . .. .. .. . . . 9.1. Eyes ............................................................... 183 9.2. Other Sense Organs.. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. 191 10. Nervous System.. , . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. 198 11. Digestive Tract ........................................................... 200 201 12. Parenchyma.............................................................. 13. Behaviour................................................................ 203 13.1. Role of the Oncomiracidium.. . . . . . . . . . . .. . . . . . . . . 203 13.2. Emergence of Oncomiracidia from Eggs . . . .. . . . . . . . . . . . . . 205 207 13.3. Swimming Behaviour.. ............................................ 13.4. Host Finding and Host Recognition . . . . . . . . . . . , . . . . . . . . . . . . . . 211 13.5. A Dispersal Phase? ................................................. 213 , . . . . . . . . . . . , . , 213 13.6. Invasion of Hosts by Oncomiracidia.. . . . . , , . 13.7. Summary ........... .............................................. , 215 14. Conclusions .........................,.............................. ...... 215 218 Acknowledgements .......................................................... References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , . . . . . . . . . . . 218

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ABSTRACT There has been no comprehensive review of the infective larval stage (oncomiracidium) in the direct life-cycle of monogeneans since Llewellyn (1963, 1968). In the last 30 years, knowledge of the general anatomy and morphology of oncomiracidia has increased significantly as has information on swimming behaviour and egg-hatching strategies that may enhance chances of host infection. Nevertheless, oncomiracidia are known for only a small proportion of monogenean species described. This review consolidates established, and summarizes new, knowledge since Llewellyn’s work and integrates light- and electron-microscopy studies including unpublished data. Currently there is considerable debate, fuelled largely by phylogenetic studies using molecular techniques, about whether or not the class Monogenea (comprising subclasses Monopisthocotylea and Polyopisthocotylea) is monophyletic. This challenges established views that Monopisthocotylea and Polyopisthocotylea form a single clade based on two larval characters: two pairs of rhabdomeric eyes; three bands of ciliated cells. In an attempt to reveal further synapomorphies for the entire Monogenea (or provide evidence against its monophyly) or possibly for the Monopisthocotylea and Polyopisthocotylea only, we review the following larval features: haptoral sclerites; ciliated cells; epidermis; terminal globule; gland, protonephridial and nervous systems; sense organs; digestive tract; parenchyma;

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and behaviour. Conclusions are equivocal but indicate that further larval studies, especially ultrastructural, are necessary to assess: the presence or absence of ‘false’ vertical rootlets of epidermal cilia; tapering epidermal cilia; the protonephridial system; the presence or absence of a terminal globule; glands and their secretions; and the embryology and chemical composition of haptoral sclerites. Future integration of light- and electron-microscopy studies are likely to be particularly informative.

1. INTRODUCTION

The Monogenea are parasitic flatworms most of which inhabit the gills, skin and fins of fish. Some have adopted a mesoparasitic way of life infecting, for example, the urinary bladders and cloaca1 bursae of turtles or the digestive tract or coelom of fish (Kearn, 1994). Monogenea are hermaphroditic and have a direct life-cycle, although circumstantial evidence suggests that two marine species of Polyopisthocotylea may have indirect life-cycles using small pelagic fish as intermediate hosts (Bychowsky and Nagibina, 1967). Because of the direct life-cycle, many species of monogeneans are of great economic importance, particularly in aquaculture. The oncomiracidium is the larval stage of monogeneans and has the task of locating, attaching and establishing itself on the host. For this reason, studies of its morphology and behaviour are important. The monograph by Bychowsky (1957, English translation 1961) contains a section on oncomiracidia and, although larvae of less than 30 species had been described at the time, it remains a useful source of information. Numerous studies on oncomiracidia were carried out between 1955 and 1963, and two excellent reviews by Llewellyn (1963, 1968) compiled information about the larvae of more than 100 species. Llewellyn (1972; see also Kearn, 1981) reviewed the behaviour of oncomiracidia. Even three decades since the last major reviews of the estimated 3000-4000 species of Monogenea that have been described (Whittington, 1998) and the 20 000 or more species that have been estimated to exist (Rohde, 1996; Whittington, 1998), only a small proportion of their larvae have been studied. Undoubtedly this paucity of information on the larvae of monogeneans relates to the relative difficulties in obtaining live adult parasites, and collecting and hatching their eggs. Even when larvae are hatched, the majority are minute ( 90%, P < 0.01) in the number of both mobile and chalimus III-IV were observed. No significant reductions were observed on the numbers of chalimus 1-11. Recent laboratory studies have confirmed efficacy of cypermethrin against all chalimus stages with a significant reduction in mean numbers (83%, P 80% were reported for Atlantic salmon fed three doses of ivermectin at 0.20 mg kg-I body weight or one dose at 0.50-1.0 mg kg-' body weight (Johnson et al., 1993b). Atlantic salmon were more susceptible to the toxic effects of ivermectin than chinook or coho, which were the most resistant. The surviving fish also showed a loss of equilibrium, reduction in feeding activity and darkened in colour. Histological examination of the major organs showed no pathological changes that could be associated with ivermectin toxicity (Johnson et al., 1993b). The LDSOfor a single oral intubation of Atlantic salmon with ivermectin was recorded as 0.5 mg kg-' and 17 pg L-' for a 96-hour immersion (Kilmartin et al., 1996). H0y et al. (1990) followed the distribution of orally intubated [3H]ivermectinat 0.2 mg kg-' body weight. They found high concentrations in the central nervous system, indicating a poorly developed blood- brain barrier in salmon compared to mammals. This may explain the relatively low margin of safety for this drug towards fish species (H0y and Horsberg, 1991). The excretion of the drug was very slow, with the treatment dose being reduced to 19% after 28 days. The drug was mainly excreted unchanged via the bile (Hay et af., 1990). Kennedy et al. (1993) treated Atlantic salmon orally with 0.05 mg kg-I body weight twice weekly for 2 months. At 0 degree days withdrawal, the concentration of ivermectin in brain, gill, kidney, skin and spleen were very similar (47-87 ng g-I). The ivermectin concentration in muscle was lower than that detected in any other tissue (35.2 ng gg'), whereas that in the liver was higher (459.8 ng g-I). The half-life ( t 1 / 2 ) of ivermectin in liver, muscle and skin ranged from 89 to 98 degree days (OD). Roth et al. (1993~)found longer retention times in the skin (t112= 188.1'0) than for the muscle ( t 1 / 2 = 120.4"D) following an oral treatment of 0.05 mg kg-' once a week for 9 weeks. Residues of 7.2 f 4.7 pg kg-I could be detected in muscle tissue following 500 "D withdrawal and in the skin following 750"D withdrawal (5.2 f 1.5 pg kg-I). No levels were detected after 1000"D withdrawal (67 days at 15°C). Environmental risks of terrestrial use of ivermectin and abermectin are usually considered to be low as the compounds are rapidly photolysed, have a high affinity to particles and are not bioaccumulated (Grant and Briggs,

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1998a). Use of ivermectin in the marine environment may lead to a longer residence time as the rate of breakdown is dependent upon the amount of light present and temperature (Grant and Briggs, 1998b). Owing to the potentially long residence time in sediment, concern has been expressed over the impact of ivermectin to benthic organisms below cages. Black et al. (1 997) found ivermectin had a significant effect on the polychaete Capitella sp. at applications of 8 1 and 8 10 pg mP2.This concentration was an order of magnitude greater than would be expected from a single treatment. The shrimp Crangon septemspinosa was unaffected by ivermectin in water at 21.5 pg L-I, but sensitive when fed on salmon pellets containing ivermectin (96-hour LD50 = 8.5 pg g-' food). The NOEC value was 2.6 pg g-' (Burridge and Haya, 1993). Although ivermectin has not been fully licensed for use in any country (Smith et al., 1993), it has been used extensively in Ireland (Costello, 1993). Small-scale trial use has been permitted at a limited number of sites by SEPA in Scotland, after research to determine ecotoxicology (Anonymous, 1996). 8.1.6. DiJlubenzuron Diflubenzuron is an established insecticide belonging to the insect growth regulator (IGR) group (Hay and Horsberg, 1991). During the intermoult period, the effect of the pesticide on crustaceans was negligible (Horsberg and H0y, 1991). The compound is registered as Dimilin@ (T. H. Agricultural & Nutrition Company) in the USA for control of the gypsy moth and in Canada for the control of larval mosquitoes (WHO, 1996). The effect against L. salmonis was examined by H0y and Horsberg (1991) who found that an oral dose of 75 mg kg-' over 14 days produced a significant reduction in adult and larval stages of sealice. This dose was considered high when compared to other compounds such as ivermectin (0.05 mg kg-' for 2 days) to achieve the same result (Roth et al., 1993a). Further trials have shown 98-100% efficacy against L. salmonis after 14 days of oral treatment at a much lower dose range of 2.26-4.54 mg kg-I, within the temperature range of 7-17°C (Erdal, 1997). It is efficacious against all developmental stages of L. salmonis, although the effect against adults is low because the moulting sequence has been completed. Some chitin deposition is still thought to occur especially during deposition of egg sacs. Efficacy is increased if treatments are targeted against the early chalimus stages and would be particularly useful in strategic management (Erdal, 1997). Diflubenzuron was poorly adsorbed by the gut of salmon with peak concentrations representing only 3.75% of the administered dose, found in

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the blood, muscle, liver and kidneys 12 hours after administration. A proportion of the compound was found to accumulate in the cartilaginous tissues (Horsberg and Hay, 1991). Despite low adsorption from the intestine, approximately 4 pg g-' of diflubenzuron was detected in the skin mucus layer 48 hours after administration. Up to 37% of the compound was rapidly metabolized and excreted via the bile within 6 hours of administration. Varying concentrations of 14C-labelled diflubenzuron (and/or metabolites) were present in the bile for 10 days, after which they began to decrease significantly. Diflubenzuron as (Lepsidon@;Ewos as.) is available for use in Norway as a formulated feed pellet. Its use is currently controlled under a conditional exemption where use is restricted to smolts in the first year at sea. A conditional exemption is granted for a 2-year period. A full market authorization licence in Norway is expected in the near future (C. Wallace, personal communication). 8.1.7. Tejlubenzuron Other IGRs are also being assessed in the control of sealice. These include teflubenzuron (Ektobannm-Skretting a s . in Norway and Calicide@in the UK). An oral treatment of 10 mg kg-' body weight daily for 7 days resulted in a reduction in lice levels of 90%, 7 days after treatment (Grantvedt, 1997). Treated sealice were attached more loosely than controls, were less mobile, a smaller size and had a slower rate of development. The cuticle lacked organization, was only half as thick and the basal membrane was absent. Greater changes were found in sealice collected from the gills than from the skin. This may be due to those on the gills being more exposed to the compound from the richer supply of blood (Grantvedt, 1997). In the control group, 90% of sexually mature females had developed egg sacs, with none being observed in the treated group. In field trials, some ovigerous females had developed prior to the treatment. The egg sacs subsequently exposed to teflubenzuron had major deformations and defects (Grantvedt, 1997). Further toxicological studies on non-target species and environmental impact assessments are currently being conducted in order to determine the viability of use of IGRs in salmon production (Ritchie, 1995). Teflubenzuron is currently in use in Norway and controlled under a 2-year conditional exemption. IGRs will have to be used in conjunction with compounds that are effective against adult stages such as pyrethroids. Their controlled, targeted use will be especially useful during periods of increased larval settlement on production sites such as spring (Wadsworth et al., 1997).

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8.2. Wrasse

Initial aquarium studies found that four species of wrasse, namely goldsinny Ctenolabrus rupestris, rock cook Ctenolabrus exoletus, corkwing Crenilabrus melops and, to a lesser extent, cuckoo Labrus mixtus removed lice from salmon (Bjordal, 1988, 1990, 1991). Wrasse have also been observed removing mobile L. salmonis and C . elongatus from salmon in production cages (Costello and Donelly, 1991; Treasurer, 1991, 1994, 1996a; Darwall et al., 1992). There was some evidence for removal of chalimus stages (Costello, 1993), although this has not been reported in other studies. In field trials, cages with wrasse stocked at 1:lOO salmon had mean lice numbers ranging from 3 to 12. Lice numbers in the controls cages rose to 46 per fish. Wrasse were transferred to these cages to avoid treating them with dichlorvos and there was an immediate reduction to 12 lice per fish. Once the wrasse were removed, the numbers of lice rose to 45 per fish within 8 days (Treasurer, 1993). The number of lice consumed by an individual goldsinny was in the range of 26-46 lice per day, wet weight 349-907 mg, representing 1.2-2.7% body weight for a fish of 30 g (Treasurer, 1993). This cleaner fish technology has been widely adopted in Norway and Scotland. By 1994 over 130 farm sites in Norway were using 1.5 million wrasse and 30 farm sites in Scotland were using 150 000 wrasse (Costello, 1996). These fish were all wild caught from inshore coastal waters (Sayer et al., 1996; Treasurer, 1996b). Concern has been expressed over the impact on local stocks if this effort continues or increases (Danvall et al., 1992; Varian et al., 1996). Long-term or widespread effects on wrasse populations are unlikely owing to their early maturity, high fecundity, abundance in areas distant from farms and capacity for rapid recruitment (Darwall et al., 1992; Costello, 1993). Preliminary trials have been conducted on culturing wrasse intensively and the initial results appear encouraging (Sherwood, 1990; Skiftesvik et al., 1996; Stone, 1996). There are difficulties associated with the use of wrasse in sealice control that have led to a reduction in the number of wrasse deployed in recent years (J.W. Treasurer, personal communication). Both typical and atypical Aeromonas salmonicida have frequently been isolated from wild-caught wrasse (Treasurer and Cox, 1991; Frerichs et al., 1992; Costello, 1993; Treasurer and Laidler, 1994; Costello et ul., 1996; Karlsbakk et al., 1996). Wrasse are susceptible to both typical and atypical strains of A . salmonicida as well as Vibrio anguillarum (serotypes 01 and 02), resulting in very high mortality of the introduced stock (Gravningen et al., 1996). Although the atypical strain was non-pathogenic to salmon (Frerichs et al., 1992), it represents a significant risk to the industry owing to the limited number of licensed antibiotics and vaccines available to combat A . salmonicida (Evelyn, 1997). There is also the potential for viral transmission from wild-caught

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wrasse (Gibson and Sommerville, 1996). However, many of these potential health risks could be alleviated by using cultured wrasse, reared in protected conditions.

8.3. Management The coordination of fallowing and the stocking of single-year class fish within designated management areas has led to a significant reduction in lice infestations in Ireland (Jackson et al., 1997), Scotland (Grant and Treasurer, 1993) and Norway (Boxaspen, 1997). Bron et al. (1993a) found that smolts introduced into a multiyear class site in April quickly became infected with lice and needed to be treated by June. Smolts introduced into a single-year class site, after the site had been fallowed, were not heavily infected with lice and no treatments were needed until the following winter. A number of sites were examined with varying length of fallow periods. It was found that the longer the fallow period, the longer the refractory period before lice numbers rise to a level where treatment becomes necessary. The numbers of C.elongutus were not influenced by a fallow period. Grant and Treasurer (1993) observed that smolts transferred to a multiyear class site were infected with copepodids of L. salmonis after 3 days and required treatment within 4 weeks of transfer. The minimum fallow period recommended was 30 days over the period February to March, although survival of females off host 32 days in the laboratory according to Grant and Treasurer (1993) should be taken into account. In 1997, 166 (49.7%) production sites in Scotland incorporated a fallow period. On the sites that did not fallow, the practice of stocking with multiple-year classes of fish was still common (SOAEFD, 1997). The increasing use of photoperiod (SOAEFD, 1997) for smolts will have to be carefully managed to ensure adequate fallow periods remain within loch systems. Understanding the epidemiology of sealice infestations in relation to these management and treatment strategies is essential for implementing effective control measures (Tully, 1989). Wootten et ul. (1982) examined the succession of generations of L. salmonis and recommended treatment at the preadult stage prior to the development of ovigerous females owing to the ineffectiveness of dichlorvos to remove larval stages. Bron et al. (1993d) examined the influence of dichlorvos treatments on the epidemiology of L. salmonis. A general rise in the intensity of infection was observed and this was temporarily interrupted by treatments. On multiyear class sites, numbers of sealice built up far more rapidly than on single-year class site, despite treatment. Where treatments were conducted against a population sensitive to dichlorvos, a large proportion of the mobile stages were removed as well as a reduction in the number of settled larval stages observed

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following treatment. It was concluded that, owing to the fall in the number of larvae following treatment, the sites were principally self-infecting. It was suggested that, in order to reduce the pool of infection for a site as a whole, all groups on a farm needed to be treated within a short space of time. At some production sites, the initial infection of L. salmonis is derived from an external source (S.L. Wadsworth, personal observation). Dispersal and transmission of infective stages of L. salmonis is not fully understood, but is likely to be influenced by a number of factors including distance, temperature and hydrography between sites (Heuch, 1995; Costelloe et al., 1996a; Boxaspen, 1997; Jackson et al., 1997). Extending treatment areas to coordinate between farms within a loch system has also increased efficacy (Wadsworth et al., 1997). By instigating a series of coordinated, synchronous, strategic treatments throughout the loch system during the winter, initial chalimus levels during the spring were significantly reduced by 90% (P< 0.001). Lice numbers for the rest of the production cycle were significantly lower (P< 0.01). There was also a reduction in the number of treatments needed, an increase in the interval between treatments, reduced fish mortalities and improvements in fish harvest quality (Wadsworth et al., 1997). Larger geographical areas and a greater number of production sites were incorporated into the treatment strategy during 1998 and coordinated by the Scottish Salmon Growers Association (SSGA). A number of these sites throughout the west coast of Scotland have shown beneficial effects (Rae, 1998). Coordination of treatments and common winter delousing has also been attempted in Norway (Melingen, 1997) with positive effects (C. Wallace, personal communication). Winter and spring delousing strategies have been based upon the seasonal variation observed at many sites in the epidemiology of L. salmonis. Low recruitment of L. salmonis during the late winter (January-early March) followed by increased larval settlement observed during the spring have been reported by a number of authors (White, 1940, 1942; Wootten et al., 1982; Hogans and Trudeau, 1989b; Jackson et af., 1997). High lice numbers have also been observed during the winter (Wootten, 1985; Bron et al., 1993c) but this may be due to milder temperatures occurring during the earlier winter periods (Boxaspen, 1997). Hogans (1995) observed that the proportion of ovigerous females to non-ovigerous females was higher towards the end of winter. The production of egg sacs by mature females was relatively constant until the end of March when a significant increase was noted. The rate of infection by copepodids only increased with rising water temperature ( r 2 = 0.82) at the end of March. Rising water temperature has an important effect upon the intensity of infection. The ratio between the period during which a cohort is spawned and during which it matures is dependent upon the direction and degree of changes in water temperature (Durban and

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Durban, 1992). From this it may be predicted that the reproductive output of L . salmonis increases in the spring and reaches a peak in early summer (Tully, 1992). The intensity and timing of this peak in output may vary between years depending upon the rate of change of temperature (Tully, 1992). Total output of planktonic stages of L . salmonis from both farmed and wild salmon in some regions of Ireland is highest during the spring and early summer (Tully and Whelan, 1993). Thus it is essential to remove ovigerous L . salmonis populations from production sites prior to this period. Other Caligid parasites such as Lepeophtheirus pectoralis exhibit distinct seasonal variability in abundance (Boxshall, 1974e). Low numbers of L . pectoralis were observed over winter and the population comprised mainly adults that had survived from the previous year. The overwintering females produced 2-3 batches of eggs in April and died soon afterwards. By May over 50% of the population comprised infective copepodids from the winter female population. The seasonal variability of Lepeophtheirus sp. may be related to the density of host populations (Boxshall, 1974e; Pemberton, 1976; Tully and Whelan, 1993). 8.4. Future Control Strategies

8.4.1. Integrated Pest Management

In recent years, there has been an increasing understanding of the epidemiology of L . salmonis, the availability of more effective compounds as well as improvement in management and coordination of treatments between sites. Further improvements could be attained with the development of an integrated pest management (IPM) scheme for L. salmonis as suggested first by Pike (1989) and, more recently, repeated by Sommerville (1995). There has been increasing concern over the development of pest resistance as well as environmental impact. Discharge has been limited over the past few years and alternatives sought to reduce organophosphate use. Recent use of insect growth regulators have proved more effective as well as more compatible with biological controls (Valentine et al., 1996). Efficacy against agricultural pests has been increased further by targeting treatments to specific periods as well as using available pesticides in rotation with other controls within an IPM scheme (Marsula and Wissel, 1994; Hall and Barry, 1995; Pickett et al., 1997). Similar IPM schemes could also be adopted for salmonid aquaculture. An ideal IPM scheme would incorporate loch fallowing, stocking of single-year class fish along with hatchery-reared wrasse. The gradual increase in lice numbers during the first year at sea would be intensively monitored and the information

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freely exchanged between relevant bodies. Targeted, planned treatments could be coordinated and synchronous between sites within the management areas. Compounds available could be used on rotation to reduce the risk of the development of resistance as well as environmental impact. It is expected that an effective adoption of an IPM scheme will reduce the overall number and increase the interval between treatments. This should also reduce any adverse environmental impact. Other stakeholders should be included in any IPM scheme, such as representatives of crustacean and wild salmonid fisheries, as well as environmental groups. Rational concerns could then be identified and strategies adapted to ameliorate any impact. 8.4.2. Husbandry Basic husbandry practices could be adapted to ameliorate L. salmonis infection. It has been found that, whilst fish were hungry, they congregated near to the surface to await feed (Huse and Holm, 1993). As they were fed, they gradually descended to greater depth where they remained whilst satiated (Fern0 et al., 1995). It has been shown that fish maintained in 0-4 m will have up to 40 times more lice than fish maintained at lower depth (Hevray et al., 1997). With the advance of recent feeding technology, such as demand feeding systems, it may be possible that lice settlement could be reduced by keeping fish well satiated and maintained at a depth below the optimum for settlement of L. salmonis. 8.4.3. StresslDisease It has been demonstrated that physiological stress such as osmoregulation significantly increase susceptibility of Atlantic salmon to infection by L. salmonis (Grimnes et al., 1996; Grimnes and Jakobsen, 1996; Dawson, 1997). Thus care should be taken to transfer smolts, which are well adapted to sea water, into sites that have been fallowed and are free of L. salmonis. Routine husbandry practices such as grading and sample weighing are likely to stress and physically damage the fish, making them more susceptible to settlement. Methods and technology could be further adapted to reduce these stressors. Additional factors will also affect susceptibility to L . salmonis, such as sexual maturity (Johnson, 1993) and disease status. Fish affected by A . salmonicida had up to 70% more L. salmonis chalimus than unaffected fish (Wadsworth, 1998). Thus immune/physiological status of the fish should be an important factor in future lice control strategies (Firth et al., 1999).

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8.4.4. Immune Modulation

The immune system of fish is capable of producing a variety of responses that are capable of protection against parasitic infections (Woo and Shariff, 1990; Woo, 1992). The potential exists for immune modulation of salmonids to lead to a reduction in susceptibility to L. salmonis (Woo, 1997). An innate response by coho salmon to L. salmonis infection was characterized by acute inflammatory response and hyperplasia. In severe cases, the chalimus were completely encapsulated and the surrounding infiltrate comprised neutrophils, macrophages, lymphocytes and necrotic tissue. No reaction, or only limited response, was observed in coho salmon injected with cortisol (Johnson and Albright, 1992b). Non-specific, innate defence mechanisms were responsible for the effective control of L. salmonis on coho salmon and suppression of these responses significantly increased the levels of infection (Johnson and Albright, 1992b). A more limited response to L. salmonis infection has also been observed for Atlantic salmon. The types of proteases collected from the mucus of Atlantic salmon infected and non-infected with L. salmonis were identical. However, mucus from the infected fish exhibited substantially higher quantities of protease activity, especially serine and metalloproteases (Firth et af., 1999). These proteases affect the production of mucus as well as innate immune reactions in fish (Firth et al., 1999). Incubation of naive control mucus with sealice extract did not show any elevation of proteases. Incubation of naive control mucus with live adult sealice significantly increased protease activity, including the production of additional proteases, which could have been derived from the sealice (Firth et a[., 1999). Arctic charr and Atlantic salmon fed iodine had lower blood cortisol levels and lower numbers of C. elongatus after challenge than fish implanted with cortisol (Mustafa and MacKinnon, 1993). Variability in lice infection between individual fish was attributed to differences in genetic make up, handling and disease status (MacKinnon, 1998). Immunosuppression by cortisol has been shown to elevate infection for a range of fish pathogens including bacteria, viruses and parasites in addition to L. salmonis (Anderson, 1990; Houghton and Matthews, 1990; Espelid et al., 1996; Bly et af., 1997). Crude antigen of L. salmonis and C. elongatus injected into rainbow trout produced antibodies that reacted to antigens of mobile chalimus and eggs of lice (Stone, 1989; Wadsworth, 1989; Grayson et al., 1991). Immunohistochemical screening of monoclonal antibodies produced to L. salmonis revealed binding to oviducts, ovaries, cuticle, haemolymph and brush border of the gut epithelium (Andrade-Salas et al., 1993; Grayson et al., 1995). Naturally infected Atlantic salmon and rainbow trout have produced antibodies to various antigens of L. salmonis, including the gut (Grayson et al., 1991). Immune activity against the gut wall of the cattle tick Boophilus

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microplus has shown some reduction in tick survival and fecundity (Opdebeeck et al., 1988; Willasden et al., 1989). Potential vaccine formulations against L. salmonis have also concentrated on gut antigens. Atlantic salmon immunized with purified gut antigens of L. salmonis showed a significant difference ( P < 0.01) in the number of ovigerous females and these sealice possessed fewer eggs per egg sac. There was no difference observed in the viability of the eggs between the groups nor was there any difference in the number of other mobile stages observed (Grayson et al., 1995). Recent investigations have concentrated on the larval stages of L. salmonis, which offer increased potential for immune control, owing to their more intimate association with host tissues than mobile stages (Pike et al., 1993a). Antigenic differences were observed between chalimus and mobile L. salmonis, with antibodies being produced to both stages in rainbow trout (Grayson et al., 1991). Survival and development of L. salmonis chalimus on salmonid species may be dependent upon either masking the epitopes of exposed antigens, especially the hold fast, or suppressing the host’s immune system. It appears that the ability to mount an effective immune response against L. salmonis is highly variable between salmonid species. Atlantic salmon and sea trout are especially vulnerable to L. salmonis infection, whilst coho and chinook salmon appear relatively resistant (Johnson and Albright, 1992a; Grimnes, 1994; Grimnes and Jakobsen, 1996; Dawson et a/., 1997). Plasmacortisol levels have been shown to increase naturally in salmonids following infection with L . salmonis with significant variation between Arctic charr, Atlantic salmon and sea trout. Highest plasmacortisol levels were observed in sea trout following infection, which induced immunosuppression (reduction of lymphocytes) and elevated levels of L. salmonis in relation to the other species (Grimnes et al., 1996). Other species of Lepeophtheirus are specifically adapted to certain host species: L. hippoglossus on halibut Hippoglossus hippoglossus (Kabata, 1979); L. pectoralis on plaice Pleuronectes platessa (Boxshall, 1974e); L. thompsoni on turbot Scophthalimus maxima; and L. europaensis on brill Scophthalimus rhombus (De Meeiis et al., 1993). This suggests that certain Lepeophtheirus species have evolved mechanisms for defeating the immune responses mounted by their respective hosts. In contrast to L. salmonis, C. elongatus has an extensive range of hosts, including some 80 species of fish world-wide (Kabata, 1979). C. elongutus was found to contain a greater number and quantity of both serine and non-serine proteases than L. salmonis. This was related to the more diverse array of biochemical substrates present in the food (Ellis et al., 1990). Potential mechanisms for defeating the immune systems of fish by sealice are not fully understood and a variety of factors may be involved. Parasitic copepods have a higher number of exocrine glands present on the dorsal

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surface than non-parasitic copepods (Bell et al., 1998). Both larval and mobile stages of L. salmonis had numerous exocrine glands that stained positive for peroxidase, although other compounds may also have been present (Bell et al., 1998). The saliva of adult, female, lone star ticks Amblyomma americana contained a cocktail of pharmacologically active compounds, e.g. immunosuppressants, analgesics, anticoagulants, antiplatelet aggregatory compounds (Bowman et al., 1996). Prostaglandin (PGE2) possesses many of these activities, which are believed to be extremely important to tick infection and survival on the host. The synthesis of prostaglandins and other eicosanoids have been reported for protozoan, trematode, cestode and nematode parasites (Bowman et al., 1996). It is not known whether L. salmonis secretions include, or are able to lead to the synthesis of prostaglandins, but elevated PGE2 levels have been observed in a limited number of Atlantic salmon sera following infection with L. salmonis (unpublished data). Isolation and characterization of eicosanoids involved in immunosuppression could lead to effective immunization of Atlantic salmon. It is unlikely that any form of immune modulation would be effective as a ‘stand-alone’ control and would need to be incorporated into an overall IPM strategy including antisealice medicines. 8.4.5. Stock Selection Since 1975, breeding programmes have been established for Atlantic salmon and rainbow trout in Norway and Scotland, Selection has focused on increased growth rates, reduced frequency of early maturation, improved flesh quality and disease resistance. The programmes have dramatically improved growth, survival and production efficiency of the aquaculture industry (Refstie, 1995; Gjedrem, 1997). There have been good results to date in the selection for resistance as fish immune responses appear to have a high heritability (Fjalestad et al., 1993; Schreck, 1996). Variable resistance by fish populations have been demonstrated to bacteria, such as Vibrio anguillarum, A . salmonicida and Renibacterium salmoninarum (Beacham and Evelyn, 1992; Marsden, 1993; Lund et al., 1995). It has been estimated that selective breeding could be expected to reduce stock mortality to furunculosis by 34% per generation and that this may correspond to increased resistance to other diseases (Gjedrem, 1997). Resistance in rainbow trout to the myxosporidian Ceratomyxa shasta varied significantly between different populations (Ibarra et al., 1994) as did brook trout to the haemoflagellate Cryptobia salmositica (Woo, 1992). Variation in settlement of Caligus elongatus has been observed between different families of Atlantic salmon and it was suggested that

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resistance to sealice might be a heritable trait. However, the mechanisms of resistance were not understood (MacKinnon et al., 1995). Higher mean numbers of C. elongatus were also observed on certain families of rainbow trout but the difference was not significant. Epidermal thickness varied between the trout families, and it was thought that a thinner epidermis could allow easier access to the dermal layers for feeding on host tissues and fluids (G. Ritchie, personal communication). Significant differences in mean numbers of L. salmonis were observed after challenge on different stocks and families of Atlantic salmon (Wadsworth, 1998).

9. ECONOMICS OF SEALICE INFECTION

Sealice were the most serious threat to salmonid aquaculture in terms of economic losses (until the appearance of infectious salmon anaemia). Even where vigilance and good fortune minimize stock losses, the regular indirect wastage from treatment will translate, during a production cycle, into significant loss of profitability. These include, depending upon farm practice and severity of infection, pre- and post-treatment loss of growth from withholding feed, and inappetance due to infection and the treatment. 9.1. Treatment Costs of Sealice Infection in Scotland

This analysis of treatment costs was contributed by Scott Peddie as an abbreviated version of Peddie (1997). It attempts to quantify the enormous cost to the Atlantic salmon cage culture industry using data from Scottish sources. Whilst every effort has been made to present an accurate picture, no single model can account for all the variables and the data used are those that we believe to be reasonably representative. Sealice control can constitute a substantial proportion of variable costs so it follows that choice of treatment is vital to profitability. Not only that, the legislative choices made by the regulatory authorities such as the SEPA and the Veterinary Medicines Directorate (VMD) can have wide-ranging implications for the economics of salmon culture in Scotland. It would be tempting to simplify the issue by comparing each treatment option based on treatment cost alone, but this would be a gross oversimplification. In reality, there are many parameters to consider that can be categorized into those relating to the composite costs of treatment and those pertaining to the savings made as a result of treatment (Figure 6). Either all or a combination of these categories can be incorporated into the

312


Losses due to side effects

COSTS

t

f

MODEOFTREATMENT

) SAVINGS

Figure 6 The relationship between treatment costs and savings for L. salmonis infections of Atlantic salmon. (Reproduced with permission from Peddie, 1997.)

following three comparative economic tools: 0 0 0

cost per fish; break-even points; treatment margins.

Each of these will be used to facilitate a financial appraisal of Cypermethrin@, hydrogen peroxide, IvermectinB, AquagardB and wrasse as methods for controlling sealice. To do this, a hypothetical farm with a 27 000 m3 capacity and a target production of 540 tonnes will be used, with fish harvested after either one or two sea winters. The figures used for cost of treatments are best estimates and might not accurately reflect actual current costs. 9.1.1. Cost per Fish

Quantifying cost per fish necessitates considering the cost of the treatment (or vaccinated/screened wrasse in the case of biological control), the cost of labour involved in treatment, the value of fish lost due to side-effects and the reduction in fish growth. Additional costs, such as oxygen costs and treatment tarpaulins for bath treatments, must also be taken into account.

313

SEALICE ON SALMONDS

As a function of actual treatment cost, labour costs and reduced fish growth subsequent to treatment, the analysis indicates that sequential hydrogen peroxide bath treatments are the most expensive, regardless of the duration of the growout cycle (Table 3). Conversely, the in-feed application of Ivermectin@will minimize the financial impact, largely due to the absence of a requirement for oxygen, tarpaulins, additional labour and periods of starvation. Moreover, although wrasse are the second least expensive form of treatment, practical experience in Scotland suggests that farmers only use them for the period subsequent to smolt transfer until the first sea winter is reached. 9.1.2. Break-even Point Cost per fish only addresses one side of the equation; it does not take into consideration the savings accrued from applying the treatment in terms of reduced mortality and increased growth. To do this, it is necessary to use break-even point analysis. The break-even point occurs when the savings associated with using a particular treatment equal the costs (Figure 7). The area to the right of the intercept represents the region where the particular treatment is cost effective, i.e. savings are greater than costs; the non-cost-effective area is where costs outweigh savings. The x-axis shows the percentage of fish mortalities attributable to sealice infection. This can be related to the savings made because of treatment. Although the former may vary between sites, it is reasonable to assume that, if they are left unchecked, sealice will cause 100% mortality. Theoretically, the x-axis of the model should also incorporate reduction in growth because of infection, but as no reliable data exist to quantify this reduction, it is excluded. The break-even point can be calculated using a formula adapted from a model developed by Lillehaug, (1989) for evaluating the cost effectiveness of

Table 3 Cost per fish (Atlantic salmon) per treatment for a 540-tonne enterprise where fish are harvested after either one or two sea winters (SW). -~

~

Treatment method Cypermethrin H202

Ivermectin Aquagard Wrasse

Cost per fish for salmon sold after 1SW (pence)

Cost per fish for salmon sold after 2SW (pence)

45.53 50.01 1.62 33.68 5.61

122.69 147.66 5.79 64.82 n.a.

314

A.W. PIKE AND S.L. WADSWORTH Saving when the site is at risk from 100% sealice-induced mortality

,

Savings

Coststsavings Area where treatment is cost-effective

costs Area where treatment is not cost-effective

*100% % Mortalities attributable to sealice infection

Figure 7 Conceptual break-even point graph used to evaluate L. salmonis treatments on Atlantic salmon. (Reproduced with permission from Peddie, 1997.)

vibriosis vaccines. It can be formalized as follows:

Labour

Treatment

7

Value per fish

costs: Hme, Wh

Ptreat Cadd

The man-hours required for the treatment method concerned Workers’ hourly wage The price of treatment over the entire cycle, or the cost of wrasse (at a ratio of 1 wrasse: 50 salmon) Additional costs such as necessary equipment, the value of fish lost due to side effects and reduced growth. For wrasse, additional costs include screening a sample of fish for pathogens, identification of pathogens, stress tests and vaccinating the population against vibriosis and furunculosis.

Savings: FCR Mno

Feed conversion ratio (averaged over the entire production cycle) The expected or actual mortality because of infection

315

SEALICE ON SALMONDS

The relative percentage efficacy for the treatment method The mean weight of fish at slaughtering The price obtained for the fish per kilogramme The price of food per kilogramme (averaged over the entire production cycle).

RPE,,, Wfish

PkkPfd

Table 4 highlights the fact that the relatively high break-even points for hydrogen peroxide in both 1- and 2-sea-winter fish, in conjunction with the lowest levels of savings for application of a treatment, make it the least economically viable option. In contrast, only 1 YOoverall stock mortality has to occur to make IvermectinB treatment financially viable. Moreover, the relatively high efficacy of the product, concomitant with low purchase costs and minimal 'additional costs', means that savings are maximized by the exclusive use of IvermectinB. Furthermore, despite the fact that CypermethrinB is the second most expensive treatment to administer, its high level of efficacy ensures that savings will be maximized by using it strategically. Usage of another bath treatment, AquagardB, results in intermediate break-even points and savings. Although wrasse become economically viable at 6% sealice-induced mortality, the savings at 100% mortality are lower for wrasse than for any other treatment. This is a function of their relatively low efficacy levels.

9.1.3.Treatment Margins Treatment margins can be utilized to evaluate the impact of treatment costs on the entire farm; the calculation used to determine these margins is: Gross margin = Output - variable Costs

(2)

Table 4 Break-even points and savings at 100% L. salmonis-induced mortality for different treatments. Treatment

Break-even point (1SW fish) (Yo)

Savings at 100% sealice induced mortality ( 1 SW fish) (€1

Break-even point (2SW fish) (X)

Savings at 100% sealice induced mortality (2SW fish) (€1

18 44

650 000 325 000

24 58

315000 275 000

1 25 6

680 000 375 000 280 000

1 23 n.a.

580 000 325 000 n.a.

Cypermethrin Hydrogen peroxide Ivermectin Aquagard Wrasse

SW

=

sea winters.

316


The output side of the equation is equal to theoretical salmon sales minus the value of fish lost due to side effects minus reduction in fish growth. In the case of sealice treatments, variable costs consist of the cost of the chemical plus labour costs plus ‘additional costs’ plus the cost of smolts. By applying this equation to the range of sealice control methods under consideration, the results obtained are presented in Table 5. The difference in margin obtained on actual production of 510 tonnes is estimated to be as much as El53 000 between hydrogen peroxide and IvermectinB for 2-seawinter fish; equivalent to 30p per kilogram of salmon produced, based on calculations made in 1997. Although the economics of sealice control will vary somewhat from site to site, the following generalizations can be made: Ivermectin is the most economical treatment on the basis of cost per fish, break-even point and treatment margins; hydrogen peroxide is the least economic treatment; Cypermethrin is relatively expensive to apply, but its high levels of efficacy make it the most financially attractive form of bath treatment available, as reflected in the break-even point analysis; Aquagard is the least expensive bath treatment in terms of cost per fish; this is also reflected in the treatment margins. However, the break-even point and savings are intermediate in relation to the other modes of lice control; wrasse appear advantageous from a financial point of view in l-seawinter fish, but their relatively low efficacy impose financial restrictions where the risk of sealice-induced mortality is high. Despite the above, it is important to appreciate the fact that the financial aspect of sealice control cannot be considered in isolation. The farmer must also take into account the simplicity of treatment application, operator safety, withdrawal period, environmental impact of usage and licensing. For

Table 5 Treatment margins (before overheads) for Atlantic salmon sold after either 1 or 2 sea winters (SW). Treatment

Hydrogen peroxide Cy permethrin Aquagard Ivermectin Wrasse

Margin before overheads $7000 Fish sold after 1SW

Fish sold after 2SW

352 370 391 483 472

333 360 422 486 n.a.

317

SEALICE ON SALMONDS

example, although expensive to use, hydrogen peroxide has the major advantage of being environmentally benign. Consequently, its use may prove economic if a premium price can be achieved for fish grown under an ‘organic’ regime. Conversely, although Ivermectin appears to be an attractive option from a financial point of view, its widespread and longterm usage is precluded in Scotland. This is for two main reasons: 0 0

the manufacturers do not support its use in aquaculture, therefore it will not be licensed for use in the marine environment; and it can only be prescribed under the ‘option cascade’, thus ensuring that Ivermectin use can only be under specific circumstances where no effective alternatives exist. Moreover, even this form of restricted use may cease when another in-feed compound is authorized.

Another approach has been taken by Sinnott (1998) who has calculated losses based on the effects of starvation, losses during treatment, mark down of harvested fish due to skin damage, losses due to secondary diseases, loss of stock and cost of treatment. The losses due to sealice on a hypothetical 764-tonne production range from a low of cX94 000 to a worst case of E200 000 per year.

10. PRIORITY AREAS

FOR FUTURE SEALICE RESEARCH

As with most parasites, establishing a laboratory maintenance system is crucial to progress. This has been achieved for both major species. What has not been established are protocols enabling the culture of larval stages on artificial substrates. It would be very useful to be able to monitor the development processes of the copepodid and chalimus larvae off-host. One of the key stages in parasite life cycles is the infective stage. Research into disrupting the life cycle is directed frequently at preventing infection and, in this respect, little is known about copepodid behaviour: where it is in the environment, how it orientates, and how it locates and identifies the preferred host. Research into the defence mechanisms of the salmonid host might exploit, or strengthen, latent defences either through selective breeding programmes [a new research programme just funded will assess the heritability and variability of resistance between different stocks of Atlantic salmon to L. salmonis (C.J. Secombes, personal communication)] or by further examination of naturally occurring resistance mechanisms, such as that identified by Johnson and Albright (1992a) for coho salmon. Understanding the general ecology of sealice is important both in the context of cage-culture populations and in wild salmonids. At present there are no mathematical models to describe the population dynamics of sealice,

318


although a new project has just been funded in Scotland to address this deficiency (G. Gettinby, personal communication). If sealice can not be prevented from establishing on the host, then the next option is to interfere with their reproductive success. This could be through the use of pest-control strategies aimed at preventing males from locating and mating with females (Pickett et al., 1997) (a new project has been funded recently to investigate this). Alternatively, reproductive output might be inhibited if some means could be found to interfere with ovarian development. Research into developing a vaccine against sealice has been in progress for many years and will continue to attract funding as the preferred option for long-term control of sealice infection in cage culture. This programme needs to be evaluated and broadened to improve its chance of success by examining the early, attached stages that may be vulnerable as already mentioned. National monitoring schemes do exist but in Scotland this is done independently by the companies themselves. Apart from national monitoring programmes being encouraged, there should be international agreement on a protocol that would allow direct comparison between countries to be possible.

ACKNOWLEDGEMENTS We wish to thank the following for their generous assistance with various parts of this review: Mark Hull, Scott Peddie, Thomas Schram, Brian Stewart.

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Index

Page numbers in italics refer to figures and tables. Acanthocotyle lobianchi, oncomiracidium attachment to host 151 hatching from egg 207 acaricides, tick control 49 AChE depression, organophosphates on salmon 293, 295 adaptive immune response, T. annulata 52 adult life span, sealice 266-7 alimentary canal, L. salmonis 280 Alphamax (Alphapharma) 299 amastigote survival in macrophage, Leishmania 17-18 to promastigote transformation in blood meal, Leishmania 21 Amblyomma, tick species 45 apoptosis, inhibition, Leishmania infected macrophage 21 Aquagard (Novartis) 292, 299, 3 15, 3 16 Atlantic salmon, sealice infections cost of control 313, 314, 316 epidemiology 270, 270- 1 cage cultured 274 L. salmonis distribution on host 241, 253 response to 291, 308 attachment monogenean oncomiracidia 149, 151, 197 and settlement, sealice on salmonids 262-4 attenuated cell line vaccine T . annulata 50, 62-4 T. parva, impossibility of producing 74-5 azamethiphos 295

behaviour, monogenean oncomiracidia 203-15 dispersal 213 emergence from eggs 205-7 ADVANCES IN PARASITOLOGY VOL 44 ISBN 0-12-031744-3

host finding and recognition 21 1 12 invasion 213-14 role 203-4, 205 summary 215 swimming behaviour 207- 1 1 behavioural effects, sealice infection 290 bilharziasis in bovines 100 blood feeding, sealice 282, 288 meal, parasite differentiation in, Leishmania 21 -2 body size and water temperature, sealice 244 BoLa restricted cytotoxic T-cells 42, 57 bovine theileriosis 43-7 background and scale of problem 43-5 carrier states 44, 48, 78 chemotherapy 50 clinical and pathological features 48-9 control measures 49-51 see also Theileria annulata; Theileria parva; Theileria sergenti break-even point, control of sealice 313- 15 Bulinus africanus group geographical distribution 113, 120-1, 123 mollusc-S. bovis association, compatibility in 125, 126, 127 snail infection experiments 116- 19 transformed prevalences, S. bovis 126 forskalii group geographical distribution 113, 120, 123 mollusc-S. bovis association, compatibility in 125, 126, 127 snail infection experiments 114-15 transformed prevalences, S. bovis 126 -

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INDEX Bulinus (continued) reticulatus group geographical distribution 113, 120, 122 mollusc- S. bovis association, compatibility in 125, 126, 127 snail infection experiments 112 transformed prevalences, S. bovis 126 truncatusltropicus group geographical distribution 113, 120, 122 mollusc-S. bovis association, compatibility in 125, 126, 127, 128 snail infection experiments 106-11 transformed prevalences, S.bovis 126 buparvaquone 50, 51

cage cultured salmonids egg sac length and number of eggs per sac, L. salmonis 256 hydrographic factors, sealice transmission 275 interaction with wild salmonids, transmission of sealice 276-9 sealice infection epidemiology 273-4 Caligus centrodonti frontal filament 264-5 on wrasse 238 Caligus clemensi 243 host range 239 Caligus curtus geographical distribution 243 mouth tube 281 Caligus elongatus 234, 235, 238 adult life span 267 copepodid stage 260, 285 distribution on host 242 epidemiology Atlantic salmon, wild 270, 27 1 cage cultured salmon 274 sea trout, wild 271, 272 frontal filament 264, 265 generation times 266 geographical distribution, farmed and wild salmonids 242-3 host range 239 morphology adults 243-4 larvae 244-5 nauplius stages, development of 259 pathological effects of infection 289 reproduction copulation 254 egg development 258 egg sac production 255 mate guarding by males 252-3 water temperature and body size 244

Caligus epidemicus, frontal filament 264- 5 Caligus longicaudatus geographical distribution 243 host range 239 Caligus orientalis 238 geographical distribution 243 host range 239 Caligus teres 235, 238 host range 239 Cullorhynchicola multitesticulatus oncomiracidia, terminal globule 166, 167 Capsala martinieri oncomiracidia, parenchyma 201, 202 carrier state, theileriosis 44, 48, 78 cell biology in the post-genome era 26-7 culture vaccine, theileriosis 42, 50, 51 cell-mediated response, Theileria T. annulata cell-mediated immunity 42, 56-9 immunopathology 59-60 T. parva 67 trophozoite and schizont 69-73 T. sergenti 78-9 chaetotaxy, polyopisthocotylean oncomiracidia 195 chalimus I-IV, sealice development 260 feeding and digestion 28 1 gut morphology 280 chemoresponsiveness hatching factors, monogenean oncomiracidia species responding to 206 monogenean oncomiracidia 21 1- 12, 21 5 sealice 285 chemotherapy bovine theileriosis 50 sealice on salmonids 292-302 diflubenzuron 30 1-2 hydrogen peroxide 295-7 ivermectin 299-301 organophosphates 292-5, 295, 304-5, 306 pyrethroids 297-9 teflubenzuron 302 chinook salmon, sealice infection epidemiology 270 response 291 cholinesterase, oncomiracidial nervous system 198 chum salmon, sealice infection epidemiology 270 Cichlidogyrus halli typicus, hooklets 149, 150

ciliary eyes, oncomiracidia 186-7

INDEX ciliated cells, oncomiracidia 153-9, 216 monopisthocotylea 154-6 polyopisthocotylea 157-9 ciliated receptors 192-6 functional morphology 196-8 monopisthocotylea 192-5 polyopisthocotylea 195-6 classical complement pathway 6, 7 Clemacotyle australis, oncomiracidium, parenchyma 202 coevolutionary process 100 coho salmon, sealice infection 291, 308 ‘coiling’ phagocytosis 8 cold chain, vaccines 51, 64 complement activation 5, 6 Leishmania, promastigote entry into mammalian host 5-7 receptors, Leishmania binding to 8-9, 10 convergence, monopisthocotyleans and polyopisthocotyleans 217, 218 copepodid stage, sealice development 260 dispersion, hydrographical effects 275 frontal filaments 264-5 gut morphology 280 sensory biology 284-6 transmission biology 261 -2 attachment and settlement 264-6 host location 262-4 vibrations, sensitivity to 263-4 copulation, sealice 254 Corridor disease see East Coast fever cost, sealice infections 237 treatment cost in Scotland, analysis 31 1 - 17 cross-immunity, T. parva Muguga and Marekibuni stocks 71-2 cross-reactivity, SPAG-I and p67 55-6, 75 cross-species immunization trial, theileriosis 65-6 crustacean parasites of fish 234-5 see also sealice cutaneous leishmaniasis 2 cypermethrin (Exis) 298, 299, 315, 316 cytokines immune response subversion, Leishmania 20, 21 immune response, T. parva schizont 72-3 T. annulata infections 58 immunopathological effects 60- I dactylogyrids 213, 214 dead vaccines, T. parva 75-6 deltamethrin 299 dendritic cells, Leishmania amastigote invasion 19

341 dichlorvos 237, 292-5 L. salmonis, effects on epidemiology 304-5 diflubenzuron (Dimilin) 301 -2 digestive system Encotyllabe chironemi 200, 201 monogenean oncomiracidia 200- 1 sealice 279-81 Dimilin (T. H. Agricultural & Nutrition Company) 301 Diplozoon paradoxum oncomiracidia eyes 188, 189 rheotaxis and host invasion 214 dispersal period, monogenean oncomiracidia 213 domus, monogenean oncomiracidia 148, 149 dorsal sensilla, oncomiracidia 197-8 East Coast fever 42, 48 eclosion, sealice 258-9 economics of sealice infection 3 1 1 - I7 egg development, sealice 258 sacs, sealice length and number of eggs per 256-8 production 255 Ektobann 284, 302 Encotyllabe chironemi, oncomiracidium digestive system 200, 201 sensory receptors 193, 194 ultrastructural study, protonephridial system 178, 180 endocrine control, sealice 284 Entobdella soleae, oncomiracidium 142-3, 145 chemotaxis 21 I epidermis 160, 161 eyes ciliary 186, 187 pigment shielded 185 glands 171, 172 grouped receptors 193 host specificity 212 Entobdella spp., glands 171, 175, 176 environmental impact abermectin 300-1 ivermectin 300- 1 organophosphates 306 azamethiphos 295 dichlorvos 294 Epicotyle torpedinis oncomiracidia, sense organs 195 epidemic viseral leishmaniasis 3 epidemiology, sealice infections 268-79 epidermal erosion, sealice on salmon 287, 288 epidermis, monogenean oncomiracidia 159-63

342 Euzetrema knoepjleri oncomiracidia, pigment-shielded eyes 185 Exis see cypermethrin experimental infection. sealice on salmonids 250- I pathophysiological effects 290- 1 intermediate host spectrum, Schisrosoma bovis 120-1, 125 prevalences, compatibility in molluscSchistosoma bovis association 104-5, 106-11, 112, 114-15, 116-19, 125 eyes, monogenean oncomiracidia 183-91 functional morphology 190- 1

fallow period, fish farm management 304 Fc receptor for IgG, macrophage infection, Leishmania 19 febrifugine 50 feeding and digestion, sealice 281 -2 pathological effects 287, 288 fish farm management, sealice control 304-6 medicines, licences for 294 flame bulbs 180, 183 formula 177-8 Monopisthocotylea 178, 180, 181 Polyopisthocotylea 181-2 freshwater, premature return to, sea trout 277 frontal filaments, sealice, copepodid stage 264-5 generation times, sealice 266 genetic studies, sealice 278-9 geographical distribution Leishmania 2 Schistosoma bovis 120-1, 124 bibliographic data 102-3 mollusc intermediate hosts 113, 121-3 sealice on salmonids 242-3 geotaxis, monogenean oncomiracida 21 I gill dwelling monopisthocotyleans 213- 14 polyopisthocotyleans 2 14 glands, monogenean oncomiracidia 169-77, 217 function 175-6 Monopisthocotylea 170-2 Polyopisthocotylea 172-5 summary 176-7 glycoinositol phospholipids (GIPLs), Leishmania amastigote 18 gp63 7, 9-10 gripus 148 gut morphology, L . salmonis 280

INDEX

Gyrodact ylidae ciliary eyes 187 spike sensilla 195, 197 viviparity 142 Haemaphysalis longicornis 78 Haemaphysalis, tick species 45 halofuginone 50 hamuli, haptoral sclerites 147, 148 haptoral sclerites, monogenean oncomiracidia 146-52, 217- 18 terminology 146-7 alternate 147-8 hatching methods, monogenean oncomiracidia 21 5 rhythm, monogenean oncomiracidia 190-1, 204, 205 Helicobacter pylori, vacuole formation 16 Heteraxinoides xanthophilis oncomiracidia, glands 173 Heterocotyle capricornensis oncomiracidia, glands 170-1 Hexabothrium appendiculatum oncomiracidia glands 174 terminal globule 167 hooklets, oncomiracidia 146, 148, 149, 151 -2 embryology 149 independent activity 149, 150 numbering of I47 host specificity Leishmania and sandfly 2, 22 monogenea 211, 212 humoral response, Theileria T . annulaia sporozoite 53-6 T . sergenti 78 husbandry, sealice control 307 Hyalomma anatolicum anatolicum 44 hydrogen peroxide 295-7, 3 13, 3 16, 3 17 hydrographical effects on copepodid dispersion 275 hydroxynapthoquinones 50

Iberian populations, Schistosoma bovis 100, 128-9, 132 IL-I0 upregulation, T . parva 72, 73 immune evasion mechanisms, Leishmania 19- 21 memory, T . parva schizont antigens 70- 1 modulation, control of sealice on salmonids 308- 1I responses T . parva 69-73 T . annulata 51-62 T . sergenti 78-9 Theileria species, comparative aspects 80, 82

INDEX immunopathology T. annulata 59-61 T. parva 72, 73 India, tropical theileriosis 44 infection and treatment, T. parva 42, 50-1, 67, 73-5 innate immune response, T. annulata 52 insect growth regulator (IGR) 301-2, 306 integrated pest management, sealice on salmonids 306-7 International Council for the Exploration of the Sea 278 ivermectin 299-301, 315, 316, 317 costs 313 January disease see East Coast fever Japan, economic impact, theileriosis 45 Kuhnia srombri, glands 174 Kuhnia sprostonae glands 174 hooklets 150 laboratory maintenance, sealice 268 lakselus see Lepeophtherius salmonis lambda-cyhalothrin 299 larval development, sealice 258-60 mechanical damage to salmonids, sealice 287-8 Leishmania life cycle 4 macrophage, interaction with 5-21 parasite persistence mechanisms 18-21 promastigote invasion and infection 5-15 promastigote to amastigote differentiation IS- 18 nomenclature, developmental stages 21 parasitophorous vacuoles, inter-species differences 16 sandfly, interaction with 21-6 blood meal, differentiation in 21-2 establishment of infection 22-3 mammalian host, transmission to 24-6 metacyclogenesis 23 -4 sequencing 26-7 see also leishmaniasis Leishmania amazonensis immune response evasion 20 parasitophorous vacuoles 16. 17 Leishmania donovani LPG 13 parasitophorous vacuoles 16 Leishmania major gp63 9-10 immune evasion 20

343 LPG 12, 13 parasitophorous vacoules 16 pPPG 14, 15 Leishmania mexicana aPPG 14 immune response evasion 20 parasitophorous vacuoles 16 SAP 14 leishmaniasis 2, 4-5, 19-20 asymptomatic infection 3 clinical manifestation, factors determining 3 control 3, 4 cutaneous 2 epidemic visceral 3 geographical distribution 2 global number of infected individuals 3 host defence system 4-5 see also complement infection establishment of 5-15 first stage 15- 16 prevalence 3 see also Leishmania leishmanolysin see gp63 Lepeophtheirus cuneifer 238 geographical distribution 243 host range 239 Lepeophtheirus hospitalis, period of infectivity 264 Lepeophtheirus pectoralis 238 frontal filament 265 mating 251, 254 period of infectivity 264 seasonal variability 306 Lepeophtheirus salmonis 234, 235, 238 adult life span 266-7 attachment to host 264 body size 244 chalimus stages I-IV, development 260 chemosensory ability 285 costs and savings of treatment 312, 314, 315

delousing strategies 305-6 developmental stages 246-9 digestive system 279-81 distribution on host 240-1 epidemiology Atlantic salmon, wild 270, 270- 1 cage cultured salmon 274 sea trout, wild 271-2, 272-3 feeding and digestion 282 freshwater survival 283-4 frontal filament 265 generation times 266 genetic differentiation, populations from wild and farmed salmonids 278-9

344 Lepeophtheirus salmonis (continued) geographical distribution, infection on wild and farmed salmonids 242-3 host immune system evasion 309 location 262-3 range 239 response to 291, 292, 308 infection, first outbreaks of 236-7 morphology adults 243-4 larvae 244-5 nauplius stages development of 259 pigmented eye spots 284, 285 pathological effects of infection adult stages 288-9, 289-90 larval stages 287-8 period of infectivity 264 precopula pairs, distribution on Atlantic salmon smolts 253 reproduction copulation 252, 254 eggs and egg sac 255, 256, 257, 258 mate guarding 252-3 pair formation 251 post-mating behaviour 254 reproductive system, structure 245, 250 sensory organs 284, 285-6 light, response to L. salmonis copepodid 263 monogenean oncomiracidia 210 see also phototaxis lipophosphoglycan (LPG) 10- 14 L. major amastigote ligand 19 parasite protection in sandfly gut 22, 23 structural analysis 12- 13 live larvae, study of monogenean 153, 154, 177 vaccines T. annulafa 42, 62-4 T. parva 73-5 loch system, coordinated treatment, sealice 305 longevity, monogenean oncomiracidia 203, 210 Lufzomyia, sandfly 3

macrophages Leishmania interaction with mammalian 5-21 parasite persistence mechanisms 18-21 promastigote invasion and establishment of infection 5- 15 promastigote to amastigote differentiation 15- 18 T. annulata, protection against 58

INDEX

mammalian host, Leishmania transmission to 24-6 mapping studies, Leishmania 26 mating, sealice 250-4 copulation 254 mate guarding 252-3 pair formation 25 1 maxadilan 26 Mediterranean Coast fever see tropical theileriosis Mediterranean populations, Schistosoma bovis 100 compatibilities, mollusc intermediate hosts 129 merozoite/piroplasm, T. annulafa 61 -2 metacyclic promastigotes, Leishmania 5, 6-7 metacyclogenesis, Leishmani, sandfly host 23-4 MHC class 11, Leishmania immune response evasion 20 MHC-restricted CTLs, immune response, T. parva schizont 70 mollusc intermediate host, Schistosoma bovis 100 host spectrum 113, 120-1, 125 see also experimental: prevalences, compatibility in molluscSchistosoma bovis association monoclonal antibodies T . annulafa,antibody-mediated sporozoitk neutralization 54 to LPG, Leishmania attachment to macrophages 11 - 12 monocyte chemoattractant protein I, Leishmania infection 17 Monogenea, oncomiracidia 140- 1 behaviour 203- 15 ciliated cells 153-9 conclusions 215-18 digestive tract 200- 1 epidermis 159-63 general morphology 142-6 glands 169-77 haptoral sclerites 146-52 host finding and recognition 21 1- 12 invasion 213- 14 nervous system 198-200 parenchyma 201-3 protonephridia 177-83 sense organs 183-98 species hatching in response to chemical factors 206 terminal globule 163-9 see also Monopisthocotylea; Polyopisthocotylea

INDEX Monopisthocotylea ciliated cells 154-6 epidermis 160, 161 eyes 184-7 general morphology 142-3, 145 glands 170-2 hooklets, TEM studies 150-1 host invasion 213-14 nervous system 198 other sense organs 192-5 protonephridial system 178-9, 180 terminal globule 164-6, 169 mucocutaneous leishmaniasis 2-3 nauplius stages, sealice development 259 pigmented eye spots 284, 285 Neoheterocotyle rhinobatidis, oncomiracidium false terminal globule and blebs 166 flame bulb 180, 181 glands 172, I73 haptor 152 parenchyma 202 pigment-shielded eyes 185-6 stained 153 nervous system, monogenean oncomiracidia 198-200 NK cells response to T. annulata 57-8 NO, macrophage anti-Theileria activity 58-9 North Mediterranean zone, Schistosoma bovis 101 geographic distribution, bibliographic data 102 mollusc hosts 113, 128 transformed prevalences 126, 127 oncomiracidia, Monogenea see Monogenea, oncomiracidia Oncorhynchus nerka, freshwater survival 283 oral treatments, sealice on salmonids 299-302 organophosphates see azamethiphos; dichlorvos; trichlorophon osmoregulation salmon, pathological effects of sealice 287 sealice 282-4 oviposition, sealice on salmonids 254-8 egg sacs length and number of eggs per sac 256-8 production 255 egg strings produced per mating, number of 255-6 female post-mating behaviour 254 oxytetracycline, infection and treatment method, T. parva 51, 74

345 p67 42-3,55, 67 cross-reactivity with SPAG-I 55-6, 75 T. parva vaccine 75-6 live delivery systems 76-7 Pacific salmon 269, 269-70 pair formation, sealice 25 1 paleobiogeographical scenario, Schistosma bovis 130-2 parasitophorous vacuole formation and microbicidal mechanisms, Leishmania 15- 17 parenchyma, monogenean onchomiracidium 201-3 parvaquone 50 pathological effects, sealice on salmon 286-92 behavioural effects 290 host response 291-2 mechanical damage 287-90 pathophysiological effects 290- 1 transmission of pathogens 292 Phlebotomus, sandfly 3 phosphoglycans (PG), Leishmania 9, 10- 15 photoperiod, sealice life cycle, effects on 267 maturation of females 260 phototaxis, sealice copepodids, host location 263 pigment-shielded eyes, monogenean oncomiracidia 183-4 directional light response 190 Monopisthocotylea 184-6 Polyopisthocotylea 187-9 pink salmon, sealice epidemiology 270 piroplasm stage, T. sergenti 78 Planorbarius meridjensis geographical distribution 113, 120, 121 transformed S. bovis prevalences 126 intrazone variability 128 mollusc-S. bovis association, compatibility in 125, 126, 127, 127-8 snail infection experiments 104-5 Plectanocotyle gurnardi oncomiracidium 144, 145-6 flame bulbs 182 glands 144, 172 pigment-shielded eyes 188 terminal globule 144, 167 Polyopisthocotylea 2 17 ciliated cells 157-9 epidermis 160, 162-3 eyes 187-90 general morphology 144, 145-6 glands 172-5 host invasion 214 nervous system 198, 199

346 Polyopisthocotylea (continued) other sense organs 195-6 protonephridia 180-2 families in which described 179 terminal globule 166-8, 168-9 species identified in 165 Polystoma integerrimum oncomiracidia epidermis 162-3 host invasion 214 Polystoma pelobatis oncomiracidia, epidermis 162-3 polystomatids, oncomiracidia eyes 188 host invasion 214 population decline, wild salmon 276-8 post-mating behaviour female sealice 254 Pricea mulrae oncomiracidia, nervous system 198, 199 procyclic promastigotes, Leishmania 21 -2 promastigotes, Leishmania entry into mammalian host 5-7 ligands for host macrophages 9-10 metacyclic and procyclic 5, 6-7 phagocytosis by macrophages 7-9 promastigote to amastigote differentiation in macrophage 15- 18 proteophosphoglycans (PPG) 14- 15, 23 protonephridia, oncomiracidia 177-83, 216 families in which described 179 Monopisthocotylea 178-9, 180 Polyopisthocotylea 179, 180-2 Protopolystoma xenopodis oncomiracidia, nervous system 198, 199 Pseudodiplorchis americanus oncomiracidia cytoplasmic connections, larvae and embryos 201 and host coevolution 204, 205 pyrethroids 297-9 ‘quorum sensing’ 23 Rajonchocotyle emarginata oncomiracidia glands 174 hatching rhythm 190 recombinant vaccines T. parva 75-6 live delivery systems 76-7 refringent droplets, oncomiracidia 200 resmethrin 299 rhabdomeric eyes, oncomiracidia Monopisthocotylea 143, 216 Polyopisthocotylea 189, 216 rheotaxis, monogenean oncomiracidia 21 1, 214 Rhipicephalus, tick species 45 rhythmic hatching, monogenean oncomiracidia 190, 204, 205

INDEX

Salartect (Solvay-Internox Ltd) 297 salinity copepodid response to 263 and survival rates, L. salmonis 283-4 salmon cage cultured see cage cultured salmonids louse seecaligus teres migration 261 production 236-7 species of sealice on 238 wild see wild salmonids Salmosan (Novartis) 295 sandfly, Leishmania host 2, 3, 5 blood meal, parasite differentiation in 21-2 establishment of infection 22-3 feeding habits and mammalian infection 5, 24-5 metacyclogenesis 23-4 promastigote transition to mammalian host 16 sandfly saliva, promastigote virulence, role in 25-6 specificity 2, 22 Schistosoma bovis 99- 133 collection of data 102-13 compatibility, mollusc-S. bovis associations 125-8 experimental intermediate host spectrum 120-1, 125 geographical distribution 101, 124 mollusc intermediate hosts 113, 121-3, 124 natural mollusc intermediate host spectrum 113 paleobiogeographical scenario 130-2 Planorbius metidjenensis see Planorbarius metidjenensis three main populations 128-30 schistosomiasis 100 schizont stage, Theileria 43-4 T. annulata 56-61 tissue culture vaccines 62-4 T. parva 42 immune responses 69-73 Scottish Salmon Strategy Task Force 277 sea trout, wild ascending Irish rivers, sealice infections 276-7 decline of populations 277 sealice infection epidemiology 268-9, 271-3 sealice 233-318 common names 235 economics of infection 3 1 1- 17 epidemiology of infections 268-79 experimental infection, problems with 250- 1 genetic studies on 278-9

INDEX

geographical distribution 242-3 history and present status of problem 236-7 host distribution on 240-2 immune system evasion 309- 10 location 262-4 range 239 identification 236 infection, natural epidemics 287 life cycles 261-8, 262-8 morphology 243-5 pathological effects 286-92 physiology 279-86 reproductive system and reproduction 245-60 research 237 priority areas for future 3 17- 18 salmonids, resistance to infection 309, 310-11 species on salmonids 238 transmission of pathogens by 292 treatment and control 292-31 1 see also Caligus elongatus; Lepeophtheirus salmonis seasonal effects, sealice 256, 267 secreted acid phosphatase (SAP), Leishmania mexicana 14 sense organs, monogenean oncomiracidia 183-98 sensilla, monogenean oncomiracidia 192, 193, 195, 197 sensory biology, sealice 284-6 silver nitrate staining, live monogenean larvae 153, 154, 192 skin parasitic monopisthocotyleans 213 snail infection experiments Bulinus africanus group 116-19 forskalii group 114- 15 reiiculaius group 112 truncatusltropicus group 106- I I Planorbarius metidjenensis 104-5 sockeye salmon 270 South Mediterranean zone, Schistosoma hovis 101, 128 geographical distribution, bibliographic data 102-3 mollusc hosts 113 transformed prevalences 126, 127 South Saharan zone, Schistosoma hovis 100, 101, 128 geographical distribution, bibliographic data 103 mollusc hosts 113, 129-30 transformed prevalences 126, 127 origins of 130

347 Southern hemisphere salmon farming 243 SPAG-I 42, 54, 55 cross-reactivity with p67 55-6, 75 subunit vaccine development 64 trials 64-6 spike sensilla, gyrodactylids 195, 197 sporozoites surface proteins see p67; SPAG-I T. annulata 45, 53-6 T. parva, immune responses 69 stock selection, control of sealice on salmonids 310-11 stress/disease, susceptibility, salmon to sealice 307 subunit vaccines T. annulaia 64-7 T . parva 42-3 T . sergenti 43 summer lesion syndrome see sealice swimming behaviour, monogenean oncomiracidia 207- 1 I , 21 5 environmental stimuli, responses to 208-9

T-c& immune response, T. parva 69-71 inappropriate activation, T. annulata 59-60 Tams1 42, 61-2 subunit vaccine development 64 trials 66-7 teflubenzuron 284, 302 temperature effects on sealice 267 egg sac length 256, 258 size 244 terminal globule, monogenean oncomiracidia 163-9 Monopisthocotylea 164-6 Polyopisthocotylea 165, 166-8 possible functions 168 species identified in 165 tetracyclines, infection and treatment method, T . parva 74 Theileria benign species, nomenclature 44-5 life cycle 45 T. anulata, T. parva, T . sergenti, comparative aspects 46-7, 80-2 Theileria annulaia 42, 46- 7 , 51 -67 immune responses 51-62 merozoite/piroplasm 61 -2 schizont 56-61 sporozoite surface proteins see SPAG-1 sporozoites 45, 53-6 trophozoite 56 vaccination 62-7 attenuated cell line vaccine 50, 62-4 subunit vaccine development 64-7 see also tropical theileriosis

348 Theileria parva 42, 44,46-7, 67-77 immune responses 69-73 sporozoite surface proteins see p67 sporozoites, donor host to recipient host transfer 45, 74-5 vaccination 73-7 live, infection and treatment method 42-3, 50-1, 67, 73-5 live, mild strains 75 recombinant vaccines 75-7 see also East Coast fever Theileria sergenti 42, 43, 46-7, 77-80 clinical features and control 48, 78 immune responses 78-9, 81 vaccination with non-living components 79-80 Theileriosis see bovine theileriosis tick control 49-50 Tonkinopsis tranfretanus oncomiracidia, glands 174 topical treatments, sealice on salmonids 292-9 Toxoplasma gondii 8 transmission biology, sealice 261 -6 treatment margins, control of sealice on salmonids 315, 316-17 trichlorophon 237 trophozoite T. annulata 56 T. parva, immune responses 69-73 tropical theileriosis 42 clinical and pathological features 48-9 Trypanosoma cruzi 8 turning sickness 49 Udonella caligorum, sense organs 192 ultrastructural studies, monogenean oncomiracidia, phylogenic implications 216- I7 uncinuli 148 Urocleidus adspectus oncomiracidia, glands 171

vaccination L. salmonis, potential development against 309

INDEX leishmaniasis control 3, 4 T. annulata 50, 62-7 T. parva 42-3, 50-1, 67, 73-7 T. sergenti 79-80 Theileria species comparative aspects 80, 82 the future 82 tick control 49-50 vibration, sensitivity to, L. salmonis copepodids 263 -4 virulence factors, Leishmania 13- 14, 25-6 visceral leishmaniasis 3 West Highland Sea Trout and Salmon Group 177 white spot see sealice wild salmonids decline of populations 276-8 egg sac length and number of eggs, L. salmonis 256 interaction with farmed salmonids, sealice transmission 276-9 sea trout, sealice infection epidemiology 268-9, 271-3 sealice infection epidemiology 268, 269-71 pathophysiological effects 290 patterns, coastal waters and high seas 26 1 World Health Organization, leishmaniasis estimates 3 world-wide salmon production 236 wrasse, control of sealice 303-4 Caligus centrodonti 238 economic viability 315, 316 Zeuxapta seriolae, anterior sensory receptors 196 Zeuxapta seriolae oncomiracidia epidermis 162, 163 glands 174, 175 parenchyma 202 pigment-shielded eyes 188, 189 protonephridial system 182 terminal globule 167, 168 ‘zipper’-type phagocytosis 8

Contents of Volumes in This Series Volume 31 Parasitic Infections in Women and their Consequences . . . . . . . . L. BRABINAND B. J . BRABIN The Pathophysiology of Malaria . . . . . . . . . . . . . . . . . N. J. WHITEAND M. HO The Interaction of Leishmania Species with Macrophages . . . . . . . J. ALEXANDER AND D. G. RUSSELL The Effects of Trypanosomatids on Insects . . . . . . . . . . . . G. A. SCHAUB Echinococcus multilocularis Infection: Immunology and Immunodiagnosis . B. GOTTSTEIN Nematodes as Biological Control Agents: Part I1 . . . . . . . . . . I. POPIEL AND w . M. HOMINICK

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83

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38 I

Volume 32 Blasfocysris in Humans and Animals: Morphology, Biology, and Epizootiology . . . . P. F. L. BOREHAM AND D. J. STENZEL Giardia and Giardiasis . . . . . . . . . . . . . . . . . . . . . . . . . J. A. REYNOLDSON AND A. H. W. MENDIS R. C. A. THOMPSON, Immunology of Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . F. Y.LIEWAND c . A. ODONNELL Transport of Nutrients and Ions across Membranes of Trypanosomatid Parasites. . . D. ZILBERSTEIN The Biology of Fish Coccidia . . . . . . . . . . . . . . . . . . . . . . A. J. DAVIESAND S. J. BALL The Sexuality of Parasitic Crustaceans . . . . . . . . . . . . . . . . . . . A. RAIBAUTAND J. P. TRILLES

I 71 161

26 1 293 367

Volume 33 The Treatment of Human African Trypanosomiasis . . . . . . . J. PEPIN AND F. MILORD Plasmodium Species Infecting Thamnomys rurilans: a Zoological Study I. LANDAU AND A. CHABAUD Metacercarial Excystment of Trematodes . . . . . . . . . . . B. FRIED The Minor Groups of Parasitic Platyhelminthes . . . . . . . . K. ROHDE Sarcoptes scabiei and Scabies. . . . . . . . . . . . . . . . . I. BURGESS

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49

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91

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235

350

CONTENTS OF VOLUMES IN THIS SERIES

Volume 34 Molecular Studies for Insect Vectors of Malaria . . . . . . . . . . . . . . . . J. M. CRAMPTON The Ribosomal RNA Genes of Plasmodium . . . . . . . . . . . . . . . . A. P. WATERS Molecular Mimicry. . . . . . . . . . . . . . . . . . . . . . . . . . . R. HALL Relationships Between Chemotherapy and Immunity in Schistosomiasis. . . . . . P. J. BRINDLEY Regulatory Peptides in Helminth Parasites . . . . . . . . . . . . . . . . . D. W. HALTON,C . SHAW, A. G. MAULEAND D. SMART Bait Methods for Tsetse Fly Control . . . . . . . . . . . . . . . . . . . . C. H. GREEN

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. 33 87

. 133 . I63 229

Volume 35 Chemotherapy of Nematode Infections of Veterinary Importance, with Special Reference to Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . GEORGEA. CONDER AND WILLIAM c . CAMPBELL Parasites as Indicators of Water Quality and the Potential Use of Helminth Transmission in Marine Pollution Studies. . . . . . . . . . . . . . . . . . . . . . . . K. MACKENZIE, H. H. WILLIAMS,B. WILLIAMS, A. H. MCVICARAND R. SIDDALL Variation in Echinococcus: Towards a Taxonomic Revision of the Genus . . . . . . R.C . A. THOMPSON,A. J . LYMBERYAND c. C . CONSTANTINE How Schistosomes Profit From the Stress Responses They Elicit in Their Hosts. . . . . MARIJKEDE JONG-BRINK Myiasis of Humans and Domestic Animals. . . . . . . . . . . . . . . . . MARTINHALLAND RICHARDWALL Parasitism and Parasitoidism in Tarsonemia (Acari: Heterostigmata) and Evolutionary Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . MAREKKALISZEWSKI, FRANCOISE ATHIAS-BINCHE AND EVERTE. LINDQUIST

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145 I77 257

335

Volume 36 Rare, New and Emerging Helminth Zoonoses. . . . . . . . . . J. D. SMYTH Population Genetics of Parasitic Protozoa and Other Microorganisms M. TIBAYRENC The Biology of Fish Haemogregarines . . . . . . . . . . . . A. J. DAVIES The Taxonomy and Biology of Philophthalmid Eyeflukes . . . . . P. M. NOLLENAND I. KANEV Human Lice and Their Management . . . . . . . . . . . . . . I. F. BURGESS Ticks and Lyme Disease . . . . . . . . . . . . . . . . . . . C. E. BENNETT

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1

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27 I

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343

351


Volume 37 Nitric Oxide and Parasitic Disease. . . . . . . . . . . . . . . . . . . . . I. A. CLARKAND K. A. ROCKETT Molecular Approaches to the Diagnosis of Onchocerciasis . . . . . . . . . . J. E. BRADLEYAND T. R. UNNASCH The Evolution of Life History Strategies in Parasitic Animals . . . . . . . . . R. POULIN The Helminth Fauna of Australasian Marsupials: Origin and Evolutionary Biology . I . BEVERIDGE AND D. M. SPRATT Malarial Parasites of Lizards: Diversity and Ecology, . . . . . . . . . . . . J . J. SCHALL

1

.

57

. I06 .

135

. 255

Volume 38 Intracellular Survival of Protozoan Parasites with Special Reference to Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi. . . . . . . . . . . . . . . . . . J. MAUEL Regulation of Infectivity of Plasmodium to the Mosquito Vector . . . . . . . . . R. E. SINDEN, G. A. BUTCHER,0. BILLKERAND s. L. FLECK Mouse-Parasite Interactions: from Gene to Population . . . . . . . . . . . . c . MOULIA,N. LE BRUNAND F. RENAUD Detection, Screening and Community Epidemiology of Taeniid Cestode Zoonoses: Cystic Echinococcosis, Alveolar Echinococcosis and Neurocysticercosis. . . . . . . P. S. CRAIG,M. T. ROGANAND J. c. ALLAN Human Strongyloidiasis. . . . . . . . . . . . . . . . . . . . . . . . . D. I. GROVE The Biology of the Intestinal Trematode Echinosroma caproni . . . . . . . . . . B. FRIED AND J. E. HUFFMAN

1

53 1 I9

169 25 1 31 1

Volume 39 1 Clinical Trials of Malaria Vaccines: Progress and Prospects . . . . . . . . . . . C. A. FACER AND M. TANNER Phylogeny of the Tissue Cyst-forming Coccidia . . . . . . . . . . . . . . . . 69 A. M. TENTERAND A. M. JOHNSON 141 Biochemistry of the Coccidia . . . . . . . . . . . . . . . . . . . . . . . G. H. COOMBS,H. DENTON,S. M. A. BROWNAND K.-W. THONG Genetic Transformation of Parasitic Protozoa . . . . . . . . . . . . . . . . 221 J. M. KELLY The Radiation-attenuated Vaccine against Schistosomes in Animal Models: Paradigm 271 for a Human Vaccine? . . . . . . . . . . . . . P. S. COULSON

352


Volume 40 Part I Cryptosporidium parvum and related genera

Natural History and Biology of Cryptosporidium parvum . . . . . . . . . . s. TZIPORIAND J. K. GRIFFITHS Human Cryptosporidiosis: Epidemiology, Transmission, Clinical Disease, Treatment, Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. K. GRIFFITHS Innate and Cell-mediated Immune Responses to Cryprosporidiurn parvum . . . . C. M. THEODOS Antibody-based Immunotherapy of Cryptosporidiosis. . . . . . . . . . . . J. H. CRABB Cryptosporidium: Molecular Basis of Host-Parasite Interaction . . . . . . . . H. WARD AND A. M. CEVALLOS Cryptosporidiosis: Laboratory Investigations and Chemotherapy . . . . . . . S. TZIPORI Genetic Heterogeneity and PCR Detection of Cryptosporidium parvum . . . . . G. WINDMER Water-borne Cryptosporidiosis: Detection Methods and Treatment Options . . . C. R. FRICKERAND J. H. CRABB

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and

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37

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87

. . 12 1 . . 151 . . 187

. . 223 . . 241

Part 2 Enterocytozoon bieneusi and Other Microsporidia

Biology of Microsporidian Species Infecting Mammals . . . . . . . . . . . . . 283 E. S. DIDIER,K. F. SNOWDENAND J. A. SHADDUCK Clinical Syndromes Associated with Microsporidiosis . . . . . . . . . . . . . . 321 D. P. KOTLERAND J. M. ORENSTEIN Microsporidiosis: Molecular and Diagnostic Aspects . . . . . . . . . . . . . . 35 1 L. M. WEIS AND C. R. VOSSBRINCK Part 3 Cyclospora cayetanensis and related species Cyclospora cayetanensis . . . . . . . . . . . . . . . Y . R. ORTEGA, C. R. STERLING AND R. H. GILMAN

399

Volume 41 Drug Resistance in Malaria Parasites of Animals and Man . . . . . . . . . . . W. PETERS Molecular Pathobiology and Antigenic Variation of Pneumocystis carinii . . . . . . Y . NAKAMURA AND M. WADA Ascariasis in China. . . . . . . . . . . . . . . . . . . . . . . . . . . PENG WEIDONG, ZHOU XIANMIN AND D.W. T. CROMPTON The Generation and Expression of Immunity to Trichinella spiralis in Laboratory Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. G. BELL Population Biology of Parasitic Nematodes: Applications of Genetic Markers . . . . T. J. C. ANDERSON, M. S. BLOUINAND R. M. BEECH Schistosomiasis in Cattle. . . . . . . . . . . . . . . . . . . . . . . . . J. DE BONT AND J. VERCRUYSSE

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63 109

149 219 285

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Volume 42 The Southern Cone Initiative Against Chagas Disease . . . . . . , . . . . , c. J. SCHOFIELD AND J. c . P. DIAS Phytomonas and Other Trypanosomatid Parasites of Plants and Fruit . . . . . , E. P. CAMARGO Paragonimiasis and the Genus Paragonimus . . . . . . . . . . . . . . . , . D. BLAIR,Z.-B. XU AND T. AGATSUMA Immunology and Biochemistry of Hymenolepis diminuta . . . . . . , . . , . . J. ANREASSEN, E. M.BENNET-JENKINS AND C. BRYANT Control Strategies for Human Intestinal Nematode Infections . . . . , . . . . . M. ALBONICO, D. W. T. CROMPTON AND L. SAVIOLI DNA Vaccines: Technology and Applications as Anti-parasite and Anti-microbial Agenis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. B. ALARCON.G. W. WAINE AND D. P. MCMANUS

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113 223

277

343

Volume 43 Genetic Exchange in the Trypunosomufidue . . . . . . . . . . . . . W. GIBSONA N D J. STEVENS The Host-Parasite Relationship in Neosporosis . . . . . . . . . . . A. HEMPHILL Proteases of Protozoan Parasites . . . . . . . . . . . . . . . . . P. J. ROSENTHAL Proteinases and Associated Genes of Parasitic Helminths . . . . . . . . J. TORT,P. J. BRINDLEY,D. KNOX,K. H. WOLFEAND J. P. DALTON Parasitic Fungi and their Interactions with the Insect Immune System . . . A. VILCINSKAS AND P. GOTZ

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Volume 44 Cell Biology of Leishmania . . . . . . . . . . . . . . E. HANDMAN Immunity and Vaccine Development in the Bovine Theilerioses. N. BOULTERAND R. HALL The Distribution of Schistosoma bovis Sonsino, 1876 in Relation Mollusc-Parasite Relationships. . . . . . . . . . . . . H. MoNE, G. MOUAHIDAND S. MOUND The Larvae of Monogenea (Platyhelminthes). . . . . . . . I. D. WHITTINGTON, L. A. CHISHOLM AND K.ROHDE Sealice on Salmonids: Their Biology and Control . . . . . . A. w.PIKE AND s. L. WADSWORTH

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